NON-ATP/CATALYTIC SITE p38 MITOGEN ACTIVATED PROTEIN KINASE INHIBITORS

ABSTRACT

Compounds that inhibit p38α MAPK protein, and methods of using the same, are provided for treating or preventing diseases such as cancer or inflammatory diseases.

This application is a continuation application of U.S. application Ser.No. 17/349,468, filed on Jun. 16, 2021, now allowed, which is acontinuation of U.S. application Ser. No. 16/872,114 filed on May 11,2020, which issued as U.S. Pat. No. 11,078,171, which is a continuationapplication under 35 U.S.C. § 111 of International Application No.PCT/US2019/064960, filed on Dec. 6, 2019, which claims the benefit under35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/776,837, filedon Dec. 7, 2018, each of which is incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The invention relates generally to compounds that are inhibitors of p38Mitogen-Activated Protein Kinases (MAPKs) proteins, and moreparticularly, but not exclusively, to compounds that inhibit p38α MAPKprotein by binding to a pocket near the ED substrate-docking site ofp38α MAPK, and methods of using such compounds as treatments fordisease.

BACKGROUND OF THE INVENTION

p38 Mitogen-Activated Protein Kinases (MAPKs) contribute to pathogenesisof many diseases, but the currently available p38 catalytic inhibitors(e.g., SB203580) are poorly effective and cause toxicity possibly due toactivity against non-inflammatory p38 isoforms (e.g., p388) and loss ofp38α-dependent counter regulatory responses (e.g., MSK1/2). Accordingly,new therapeutics and methods of treatment are needed in the field bothto address selective inhibition of p38α MAPK and to selectively blockcertain p388a MAPK functions to preserve critical counterregulatory andhomeostatic functions with application for the treatment of inflammatoryand oncologic diseases.

SUMMARY OF THE INVENTION

The disclosure provides a compound of Formula A, or a pharmaceuticallyacceptable salt, solvate, hydrate, cocrystal, or prodrug thereof:

wherein in Formula A: Ar and Ar¹ are independently selected from a mono-or polycyclic optionally substituted cycloalkyl, mono- or polycyclicoptionally substituted heterocycloalkyl, mono- or polycyclic optionallysubstituted aryl, mono- or polycyclic optionally substituted arylalkyl,mono- or polycyclic optionally substituted heteroaryl, and mono- orpolycyclic optionally substituted heteroarylalkyl; R¹ and R² areindependently selected from hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substituted alkylheteroaryl,wherein R¹ and R² can optionally be joined to form a carbo- orheterocycle; L¹ and L² are linkers comprising independently one or moreof a bond, —NR^(a)—, —S—, —S(O)—, —S(O)₂—, —O—, —CR^(a) ₂—, —C(O)O—,—OC(O)—, —C(O)S—, —SC(O)—, —C(O)NR^(a)—, —NR^(a)C(O)—, —C(O)NR^(a)SO₂—,—SO₂NR^(a) C(O)—, —OC(O)O—, —OC(O)S—, —SC(O)O—, —OC(O)NR^(a)—,—NR^(a)C(O)O—, —S(O)_(t)N(R^(a))— (where t is 1 or 2),—N(R^(a))S(O)_(t)— (where t is 1 or 2), disubstituted alkyl,disubstituted heteroalkyl, disubstituted alkenyl, disubstituted alkynyl,disubstituted cycloalkyl, disubstituted heterocycloalkyl, disubstitutedaryl, disubstituted arylalkyl, disubstituted heteroaryl, anddisubstituted heteroarylalkyl; wherein any optional substituent isindependently selected at each occurrence from optionally substitutedalkyl, optionally substituted heteroalkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionallysubstituted aryl, optionally substituted arylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —SC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a),—C(O)OR^(a), —C(O)SR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,—N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), and PO₃(R^(a))₂; and R^(a) isindependently selected at each occurrence from hydrogen, optionallysubstituted alkyl, optionally substituted fluoroalkyl, optionallysubstituted carbocyclyl, optionally substituted carbocyclylalkyl,optionally substituted aryl, optionally substituted aralkyl, optionallysubstituted heterocycloalkyl, optionally substitutedheterocycloalkylalkyl, optionally substituted heteroaryl, and optionallysubstituted heteroarylalkyl. In some embodiments, an optionalsubstituent is independently selected from R³, R⁴, R⁵, and R⁶, which areindependently selected from hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substituted alkylheteroaryl,wherein any two of R¹, R², R³, R⁴, R⁵, and R⁶ can optionally be joinedto form a carbo- or heterocycle. In some embodiments, Ar¹ is anoptionally substituted aryl or optionally substituted heteroaryl ring.In some embodiments, Ar is a mono- or polycyclic optionally substitutedaryl, a mono- or polycyclic optionally substituted arylalkyl, a mono- orpolycyclic optionally substituted heteroaryl, or a mono- or polycyclicoptionally substituted heteroarylalkyl. In some embodiments, Ar is a 5or 6 membered optionally substituted aryl or a 5 or 6 memberedoptionally substituted heteroaryl, wherein the L¹ and L² are connectedto Ar in a 1,2, 1,3, or 1,4 substitution pattern. In some embodiments,Ar is 1,2 disubstituted, 1,3 disubstituted, or 1,4 disubstituted phenyl,pyridine, pyrimidine, pyrazine, or triazine. In some embodiments, Ar is1,2 disubstituted or 1,3 disubstituted furan, thiophene, pyrrole,thiazole, imidazole, oxazole, triazole, or pyrazole. In someembodiments, Ar is 1,2 disubstituted, 1,3 disubstituted, 1,4disubstituted, 1,5 disubstituted, 1,6 disubstituted, 1,7 disubstituted,or 1,8 disubstituted naphthalene, quinoline, isoquinoline, orquinazoline. In some embodiments, Ar is 1,2 disubstituted, 1,3disubstituted, 1,4 disubstituted, 1,5 disubstituted, 1,6 disubstituted,or 1,7 disubstituted indole or imidazole. In some embodiments, L¹ is alinker selected from —CH₂—, —C(CH₃)₂—, and —C(CH₂CH₂)—; L² is a linkerselected from —NHCH₂—, —CH₂NH—, —NHCO—, —CONH—, —SO₂NH—, and —NHSO₂—;and —NR¹R² is selected from:

wherein R⁷ is hydrogen or an optionally substituted alkyl, and Ar² is aheterocycle.

In one aspect, the disclosure relates to a compound of Formula I, or apharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof:

wherein in Formula I: each of R¹, R², R³, R⁴, R⁵, and R⁶ isindependently selected from hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substituted alkylheteroaryl,wherein R¹ and R² can optionally be joined to form a carbo- orheterocycle; L¹ is a linker selected from —CH₂—, —C(CH₃)₂—, and—C(CH₂CH₂)—; L² is a linker selected from —NHCH₂—, —CH₂NH—, —NHCO—,—CONH—, —SO₂NH—, and —NHSO₂—; and Ar¹ is an optionally substituted arylor optionally substituted heteroaryl ring. In some embodiments, the—NR¹R² group in Formula I is selected from:

wherein R⁷ is hydrogen or an optionally substituted alkyl, and Ar² is aheterocycle. In some embodiments, the —NR¹R² group in Formula I isselected from:

In some embodiments, L¹ is —CH₂—. In some embodiments, R³, R⁴, R⁵, andR⁶ are hydrogens. In some embodiments, L² is selected from —NHCH₂—,—NHCO—, and —NHSO₂—. In some embodiments, Ar¹ is selected from:

wherein each of R⁷, R⁸, R⁹, R¹⁰, R, R¹², and R¹³ is independentlyselected from hydrogen, halogen, —NR¹R², alkoxy, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substituted alkylheteroaryl,wherein any two vicinal of R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³ canoptionally be joined to form a carbo- or heterocycle. In someembodiments, Ar¹ is selected from:

In another aspect, the disclosure relates to a compound of Formula II,wherein the compound is of any of Formulas 1001-1180 disclosed in Table1:

In another aspect, the disclosure relates to a compound of any ofFormulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035(SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), and 1087 (SF-7-044):

In another aspect, the disclosure relates to a compound of any ofFormulas A, I, II, and 1001-1180, wherein the compound is a p38α MAPKinhibitor. In another aspect, the disclosure relates to a compound ofany of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035(SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087(SF-7-044), wherein the compound is a p38α MAPK inhibitor. In someembodiments, the p38α MAPK inhibitor is a p38α MAPK selective inhibitor.

In another aspect, the disclosure relates to a p38α MAPK inhibitor ofany of Formulas A, I, II, and 1001-1180, wherein the p38α MAPK inhibitoris capable of binding to a pocket near the ED substrate-docking site ofp38α MAPK and defined at least by residues R49, H107, L108, and K165 inp38α MAPK. In some embodiments, the binding pocket is defined byresidues R49, H107, L108, M109, G110, A157, V158, E163, L164, and K165in p38α MAPK. In some embodiments, p38α MAPK inhibitor is a p38α MAPKselective inhibitor.

In another aspect, the disclosure relates to a p38α MAPK inhibitor ofany of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035(SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087(SF-7-044), wherein the p38α MAPK inhibitor is capable of binding to apocket near the ED substrate-docking site of p38α MAPK and defined atleast by residues R49, H107, L108, and K165 in p38α MAPK. In someembodiments, the binding pocket is defined by residues R49, H107, L108,M109, G110, A157, V158, E163, L164, and K165 in p38α MAPK. In someembodiments, p38α MAPK inhibitor is a p38α MAPK selective inhibitor.

In one aspect, the disclosure relates to a pharmaceutical compositionincluding a compound of any of Formulas A, I, II, and 1001-1180, or apharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and a physiologically compatible carrier medium,wherein the amount of compound in the composition is a therapeuticallyeffective amount for the treatment or prevention of a disease alleviatedby inhibiting p38α MAPK activity in a patient in need thereof. Inanother aspect, the disclosure relates to a pharmaceutical compositionincluding a compound of any of Formulas 1001 (SF-6-221), 1032(SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037(SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085(SF-6-222), SF-7-009, and 1087 (SF-7-044), or a pharmaceuticallyacceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and aphysiologically compatible carrier medium, wherein the amount ofcompound in the composition is a therapeutically effective amount forthe treatment or prevention of a disease alleviated by inhibiting p38αMAPK activity in a patient in need thereof. In some embodiments, thedisease is cancer or an inflammatory disease. In some embodiments, thedisease is selected from the group consisting of rheumatoid arthritis, acardiovascular disease, multiple sclerosis, inflammatory bowel disease,chronic obstructive pulmonary disease (COPD), asthma, acute respiratorydistress syndrome (ARDS), and acute lung injury (ALI). In someembodiments, the cancer is selected from the group consisting ofacoustic neuroma, adenocarcinoma, angiosarcoma, astrocytoma, basal cellcarcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breastcancer, bronchogenic carcinoma, cervical cancer, chordoma,choriocarcinoma, colon cancer, colorectal cancer, craniopharyngioma,cystadenocarcinoma, embryonal carcinoma, endotheliocarcinoma,ependymoma, epithelial carcinoma, esophageal cancer, Ewing's tumor,fibrosarcoma, gastric cancer, glioblastoma multiforme, glioma, head andneck cancer, hemangioblastoma, hepatoma, kidney cancer, leiomyosarcoma,liposarcoma, lung cancer, lymphangioendotheliosarcoma,lymphangiosarcoma, medullary carcinoma, medulloblastoma, melanoma,meningioma, mesothelioma, myxosarcoma, nasal cancer, neuroblastoma,oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer,pancreatic cancer, papillary adenocarcinoma, papillary carcinoma,pinealoma, prostate cancer, rabdomyosarcoma, rectal cancer, renal cellcarcinoma, retinoblastoma, sarcoma, sebaceous gland carcinoma, seminoma,skin cancer, squamous cell carcinoma, stomach cancer, sweat glandcarcinoma, synovioma, testicular cancer, small cell lung carcinoma,throat cancer, uterine cancer, Wilm's tumor, blood cancer, acuteerythroleukemic leukemia, acute lymphoblastic B-cell leukemia, acutelymphoblastic T-cell leukemia, acute lymphoblastic leukemia, acutemegakaryoblastic leukemia, acute monoblastic leukemia, acutemyeloblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocytic leukemia, acute promyelocytic leukemia, acuteundifferentiated leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, hairy cell leukemia, multiple myeloma, heavy chaindisease, Hodgkin's disease, multiple myeloma, non-Hodgkin's lymphoma,polycythemia vera, and Waldenström's macroglobulinemia.

In one aspect, the disclosure relates to a method of treating orpreventing a disease alleviated by inhibiting the p38α MAPK protein in apatient in need thereof, including administering to the patient atherapeutically effective amount of a p38α MAPK inhibitor, or apharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, wherein the p38α MAPK inhibitor is a compound of any ofFormulas A, I, II, and 1001-1180. In another aspect, the disclosurerelates to a method of treating or preventing a disease alleviated byinhibiting the p38α MAPK protein in a patient in need thereof, includingadministering to the patient a therapeutically effective amount of ap38α MAPK inhibitor, or a pharmaceutically acceptable salt, solvate,hydrate, cocrystal, or prodrug thereof, wherein the p38α MAPK inhibitoris a compound of any of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034(SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043(SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009,and 1087 (SF-7-044). In some embodiments, the p38α MAPK inhibitor isadministered in a dosage unit form. In some embodiments, the dosage unitincludes a physiologically compatible carrier medium. In someembodiments, the disease is cancer or an inflammatory disease. In someembodiments, the disease is selected from the group consisting ofrheumatoid arthritis, a cardiovascular disease, multiple sclerosis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), asthma, acute respiratory distress syndrome (ARDS), and acutelung injury (ALI). In some embodiments, the cancer is selected from thegroup consisting of acoustic neuroma, adenocarcinoma, angiosarcoma,astrocytoma, basal cell carcinoma, bile duct carcinoma, bladdercarcinoma, brain cancer, breast cancer, bronchogenic carcinoma, cervicalcancer, chordoma, choriocarcinoma, colon cancer, colorectal cancer,craniopharyngioma, cystadenocarcinoma, embryonal carcinoma,endotheliocarcinoma, ependymoma, epithelial carcinoma, esophagealcancer, Ewing's tumor, fibrosarcoma, gastric cancer, glioblastomamultiforme, glioma, head and neck cancer, hemangioblastoma, hepatoma,kidney cancer, leiomyosarcoma, liposarcoma, lung cancer,lymphangioendotheliosarcoma, lymphangiosarcoma, medullary carcinoma,medulloblastoma, melanoma, meningioma, mesothelioma, myxosarcoma, nasalcancer, neuroblastoma, oligodendroglioma, oral cancer, osteogenicsarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma,papillary carcinoma, pinealoma, prostate cancer, rabdomyosarcoma, rectalcancer, renal cell carcinoma, retinoblastoma, sarcoma, sebaceous glandcarcinoma, seminoma, skin cancer, squamous cell carcinoma, stomachcancer, sweat gland carcinoma, synovioma, testicular cancer, small celllung carcinoma, throat cancer, uterine cancer, Wilm's tumor, bloodcancer, acute erythroleukemic leukemia, acute lymphoblastic B-cellleukemia, acute lymphoblastic T-cell leukemia, acute lymphoblasticleukemia, acute megakaryoblastic leukemia, acute monoblastic leukemia,acute myeloblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocytic leukemia, acute promyelocytic leukemia, acuteundifferentiated leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, hairy cell leukemia, multiple myeloma, heavy chaindisease, Hodgkin's disease, multiple myeloma, non-Hodgkin's lymphoma,polycythemia vera, and Waldenström's macroglobulinemia. In someembodiments, the compound selectively inhibits p38α MAPK. In someembodiments, inhibition of p38α MAPK does not result in loss of ap38α-dependent counter-regulatory response. In some embodiments, thep38α-dependent counter regulatory-response relates to mitogen- andstress-activated protein kinase-1 (MSK1), or MSK2. In some embodiments,inhibiting p38α MAPK stabilizes an endothelial or epithelial barrierfunction. In some embodiments, inhibiting p38α MAPK reducesinflammation. In some embodiments, inhibiting p38α MAPK mitigates lunginjury. In some embodiments, inhibiting p38α MAPK mitigates LPS-inducedlung injury. In some embodiments, inhibiting p38α MAPK regulatesleukocyte trafficking. In some embodiments, inhibiting p38α MAPKregulates cytokine expression.

In one aspect, the disclosure relates to a method of treating orpreventing a disease alleviated by inhibiting the p38α MAPK protein in apatient in need thereof, including administering to the patient atherapeutically effective amount of a composition including a compoundof any of Formulas A, I, II, and 1001-1180, or a pharmaceuticallyacceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and aphysiologically compatible carrier medium. In another aspect, thedisclosure relates to a method of treating or preventing a diseasealleviated by inhibiting the p38α MAPK protein in a patient in needthereof, including administering to the patient a therapeuticallyeffective amount of a composition including a compound of any ofFormulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035(SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087(SF-7-044), or a pharmaceutically acceptable salt, solvate, hydrate,cocrystal, or prodrug thereof, and a physiologically compatible carriermedium. In some embodiments, the composition is administered in a dosageunit form. In some embodiments, the dosage unit includes aphysiologically compatible carrier medium. In some embodiments, thedisease is cancer or an inflammatory disease. In some embodiments, thedisease is selected from the group consisting of rheumatoid arthritis, acardiovascular disease, multiple sclerosis, inflammatory bowel disease,chronic obstructive pulmonary disease (COPD), asthma, acute respiratorydistress syndrome (ARDS), and acute lung injury (ALI). In someembodiments, the cancer is selected from the group consisting ofacoustic neuroma, adenocarcinoma, angiosarcoma, astrocytoma, basal cellcarcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breastcancer, bronchogenic carcinoma, cervical cancer, chordoma,choriocarcinoma, colon cancer, colorectal cancer, craniopharyngioma,cystadenocarcinoma, embryonal carcinoma, endotheliocarcinoma,ependymoma, epithelial carcinoma, esophageal cancer, Ewing's tumor,fibrosarcoma, gastric cancer, glioblastoma multiforme, glioma, head andneck cancer, hemangioblastoma, hepatoma, kidney cancer, leiomyosarcoma,liposarcoma, lung cancer, lymphangioendotheliosarcoma,lymphangiosarcoma, medullary carcinoma, medulloblastoma, melanoma,meningioma, mesothelioma, myxosarcoma, nasal cancer, neuroblastoma,oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer,pancreatic cancer, papillary adenocarcinoma, papillary carcinoma,pinealoma, prostate cancer, rabdomyosarcoma, rectal cancer, renal cellcarcinoma, retinoblastoma, sarcoma, sebaceous gland carcinoma, seminoma,skin cancer, squamous cell carcinoma, stomach cancer, sweat glandcarcinoma, synovioma, testicular cancer, small cell lung carcinoma,throat cancer, uterine cancer, Wilm's tumor, blood cancer, acuteerythroleukemic leukemia, acute lymphoblastic B-cell leukemia, acutelymphoblastic T-cell leukemia, acute lymphoblastic leukemia, acutemegakaryoblastic leukemia, acute monoblastic leukemia, acutemyeloblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocytic leukemia, acute promyelocytic leukemia, acuteundifferentiated leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, hairy cell leukemia, multiple myeloma, heavy chaindisease, Hodgkin's disease, multiple myeloma, non-Hodgkin's lymphoma,polycythemia vera, and Waldenström's macroglobulinemia. In someembodiments, the compound selectively inhibits p38α MAPK. In someembodiments, inhibition of p38α MAPK does not result in loss of ap38α-dependent counter-regulatory response. In some embodiments, thep38α-dependent counter regulatory-response relates to mitogen- andstress-activated protein kinase-1 (MSK1), or MSK2. In some embodiments,inhibiting p38α MAPK stabilizes an endothelial or epithelial barrierfunction. In some embodiments, inhibiting p38α MAPK reducesinflammation. In some embodiments, inhibiting p38α MAPK mitigates lunginjury. In some embodiments, inhibiting p38α MAPK mitigates LPS-inducedlung injury. In some embodiments, inhibiting p38α MAPK regulatesleukocyte trafficking. In some embodiments, inhibiting p38α MAPKregulates cytokine expression.

In one aspect, the disclosure relates to a therapeutically effectiveamount of a p38α MAPK inhibitor, or a pharmaceutically acceptable salt,solvate, hydrate, cocrystal, or prodrug thereof, for use in treating orpreventing a disease alleviated by inhibiting the p38α MAPK protein in apatient in need thereof, wherein the p38α MAPK inhibitor is a compoundof any of Formulas A, I, II, and 1001-1180. In another aspect, thedisclosure relates to a therapeutically effective amount of a p38α MAPKinhibitor, or a pharmaceutically acceptable salt, solvate, hydrate,cocrystal, or prodrug thereof, for use in treating or preventing adisease alleviated by inhibiting the p38α MAPK protein in a patient inneed thereof, wherein the p38α MAPK inhibitor is a compound of any ofFormulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035(SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), and 1087 (SF-7-044). Insome embodiments, the p38α MAPK inhibitor is administered in a dosageunit form. In some embodiments, the dosage unit includes aphysiologically compatible carrier medium. In some embodiments, thedisease is cancer or an inflammatory disease. In some embodiments, thedisease is selected from the group consisting of rheumatoid arthritis, acardiovascular disease, multiple sclerosis, inflammatory bowel disease,chronic obstructive pulmonary disease (COPD), asthma, acute respiratorydistress syndrome (ARDS), and acute lung injury (ALI). In someembodiments, the cancer is selected from the group consisting ofacoustic neuroma, adenocarcinoma, angiosarcoma, astrocytoma, basal cellcarcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breastcancer, bronchogenic carcinoma, cervical cancer, chordoma,choriocarcinoma, colon cancer, colorectal cancer, craniopharyngioma,cystadenocarcinoma, embryonal carcinoma, endotheliocarcinoma,ependymoma, epithelial carcinoma, esophageal cancer, Ewing's tumor,fibrosarcoma, gastric cancer, glioblastoma multiforme, glioma, head andneck cancer, hemangioblastoma, hepatoma, kidney cancer, leiomyosarcoma,liposarcoma, lung cancer, lymphangioendotheliosarcoma,lymphangiosarcoma, medullary carcinoma, medulloblastoma, melanoma,meningioma, mesothelioma, myxosarcoma, nasal cancer, neuroblastoma,oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer,pancreatic cancer, papillary adenocarcinoma, papillary carcinoma,pinealoma, prostate cancer, rabdomyosarcoma, rectal cancer, renal cellcarcinoma, retinoblastoma, sarcoma, sebaceous gland carcinoma, seminoma,skin cancer, squamous cell carcinoma, stomach cancer, sweat glandcarcinoma, synovioma, testicular cancer, small cell lung carcinoma,throat cancer, uterine cancer, Wilm's tumor, blood cancer, acuteerythroleukemic leukemia, acute lymphoblastic B-cell leukemia, acutelymphoblastic T-cell leukemia, acute lymphoblastic leukemia, acutemegakaryoblastic leukemia, acute monoblastic leukemia, acutemyeloblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocytic leukemia, acute promyelocytic leukemia, acuteundifferentiated leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, hairy cell leukemia, multiple myeloma, heavy chaindisease, Hodgkin's disease, multiple myeloma, non-Hodgkin's lymphoma,polycythemia vera, and Waldenstrom's macroglobulinemia. In someembodiments, the compound selectively inhibits p38α MAPK. In someembodiments, inhibition of p38α MAPK does not result in loss of ap38α-dependent counter-regulatory response. In some embodiments, thep38α-dependent counter regulatory-response relates to mitogen- andstress-activated protein kinase-1 (MSK1), or MSK2. In some embodiments,inhibiting p38α MAPK stabilizes an endothelial or epithelial barrierfunction. In some embodiments, inhibiting p38α MAPK reducesinflammation. In some embodiments, inhibiting p38α MAPK mitigates lunginjury. In some embodiments, inhibiting p38α MAPK mitigates LPS-inducedlung injury. In some embodiments, inhibiting p38α MAPK regulatesleukocyte trafficking. In some embodiments, inhibiting p38α MAPKregulates cytokine expression.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofembodiments of the invention, will be better understood when read inconjunction with the appended drawings and figures.

FIGS. 1A-1F illustrate the design of substrate-selective p38 inhibitors.FIG. 1A illustrates the structure of p38α showing CD, ED, DEF andactivation site. FIG. 1B illustrates the comparison between the p38α andβ structures; CD and ED sites are colored red and blue, and the CADDtarget yellow. Sequence comprising the CADD target on p38α andcorresponding site on p38β differ in only three of ten amino acids(highlighted yellow). FIG. 1C illustrates the overlap of CADD targetstructure in apo- (PDB:1P38; green) and dual-phosphorylated (PDB:3PY3;yellow) mouse p38α. FIG. 1D illustrates the overview of CADD screeningstrategy. FIG. 1E illustrates the DSF screening of compounds added at10, 25, 50, or 100 μM to recombinant p38α or ERK2 with binding indicatedby increase in melting temperature. Compounds binding ERK2 and p38 αhighlighted yellow. Those only binding to p38 α are highlighted in blue.FIG. 1F illustrates the chemical structure of UM60, UM101, and SB203580.

FIGS. 2A-2D illustrate the biological effects of p38 inhibitors. FIG. 2Aand FIG. 2B illustrate the effect of 10 μM SB203580 (SB), or indicatedconcentration of UM60 or 101 on HMVECL permeability (FIG. 2A) andcapacity for IL-8-directed neutrophil TEM (FIG. 2B). Cells werepretreated with DMSO or compounds for 1 h, then incubated with 10 ng/mlTNFα for 6 h prior to permeability assay (FIG. 2A) or at 39.5° C.without additional stimulus for 6 h prior to TEM assay (FIG. 2B).Mean±SE. * denotes p<0.0001 vs. DMSO, †p<0.0001 vs. SB, ‡ p<0.005 vs.37° C. FIG. 2C and FIG. 2D. Male CD1 mice were pretreated with 1 mg SBor 0.1-1 mg UM101 prior to i.t. instillation of 50 μg LPS andhyperthermia exposure. * denotes p<0.05 vs. DMSO.

FIGS. 3A-3K illustrate the biochemical effects of substrate-selectivep38 inhibitors. FIG. 3A and FIG. 3B illustrate the heat maps from RNASeqshowing IPA pathways inhibited by SB203580 alone or SB203580 and UM101or (FIG. 3A) and those only inhibited by UM101 (FIG. 3B). FIG. 3Cillustrate the biochemical effects of substrate-selective p38 inhibitorson HeLa cells pre-treated with 50 μM UM101 or 10 μM SB203580 (SB) for 30min, then treated with anisomycin for 10-60 min, and immunoblotted forphospho-MK2, Stat-1 and total p38. FIG. 3D illustrates the DSF analysisof UM101 and SB203580 (SB) binding to recombinant p38α and p380. Mean±SEof 4 experiments. *, †, and § denote p<0.0001 vs. p388a with DMSO, p380with DMSO, and p38p with SB203580, respectively. P<0.0001 for differencebetween UM101 binding to p38α and p38p by MANOVA. FIG. 3E illustratesthe DSF analysis of UM101 and SB203580 (SB) binding to recombinantwild-type p388a and a p388a mutant with 4 mutations in CADD-targetedpocket. Mean±SE of 4 experiments. * and t, denote p<0.0001 vs. wild-typewith DMSO and mutant with DMSO, respectively. P<0.0001 for differencebetween UM101 binding to wild-type and mutant p38α by MANOVA. FIGS.3F-3K illustrate STD-NMR performed with UM101 and p38α (FIG. 3F and FIG.3G), p380 (FIG. 3H and FIG. 3I), and the p38α mutant (FIG. 3J and FIG.3K). The 1D (FIG. 3F, FIG. 3H, and FIG. 3J) and STD spectra (FIG. 3G,FIG. 3I, and FIG. 3K) from the same sample are shown. The tentative peakassignments are indicated in FIG. 3F. The structure of UM101 with theprotons labeled is shown in the insert.

FIG. 4 illustrates the preliminary analysis of IL-8 and IL-1ß mRNA byqRT-PCR prior to RNASeq. HMVECLs were preincubated with 0.4% DMSO, 10 μMSB203580, or 100 μM UM101 for 1 h, then stimulated with 10 ng/ml TNFαfor 4 h and total RNA was collected, reverse transcribed, analyzed byqRT-PCR and fold change relative to unstimulated control cellscalculated using the delta-delta method and GAPDH as the housekeepinggene.

FIG. 5 is a quadrant map of RNASeq analysis. Genes with at least 10reads in one sample per set and at least 2-fold increase with TNFα areshown. The key refers to the direction of change in UM101-treatedcells/SB203580-treated cells vs. DMSO-treated cells.

FIG. 6 is the SILCS FragMap of p38α. Nonpolar maps (green) indicateputative binding pockets, with the location of the ED site indicated.H-bond donor (blue) and acceptor (red) maps are shown.

FIG. 7 illustrates compound UM101 overlaid on SILCS FragMaps shown inwireframe at contours of −1.0 kcal/mol for aromatic (purple), aliphatic(green), positive (cyan), H-bond acceptor (red) and H-bond donor (blue)functional groups on the p38α backbone, with sidechains of the fourresidues mutated in our CADD-site-disrupted mutant indicated. Spatialdistributions of FragMaps indicate where respective functional groupsmake favorable contributions to binding.

FIG. 8 illustrates that UM101 and analogues inhibit thrombin-inducedcell permeability. Endothelial cells were pretreated with or without 10or 15 mM of UM101, SF-6-222 (6222), or SF-7-009 (7009).Transepithelial/transendothelial electrical resistance (TEER) was usedto measure cell permeability.

FIG. 9 illustrates that UM101 and analogues inhibit acute lung injury ina mouse model. Mice were pretreated i.p. with 1 mg of test compounds andthen treated with 100 mg LPS i.t. at 39° C. to induce inflammation. Lunginjury was assessed by measuring total protein in the lung lavage fluid.

FIG. 10 illustrates that UM101 and analogues inhibit acute lung injuryin a mouse model. All mice received 50 μg LPS i.t. and were kept 24 h at39° C.; all treatments 1 mg in 0.5 ml DMSO given 6 h post-LPS; mean±SM,n=5, * p=0.03 vs DMSO #1; † p=0.16 vs DMSO #2. Lung injury was assessedby measuring total protein in the lung lavage fluid.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

Definitions

As used herein, the terms “administer,” “administration” or“administering” refer to (1) providing, giving, dosing, and/orprescribing by either a health practitioner or his authorized agent orunder his or her direction according to the disclosure; and/or (2)putting into, taking or consuming by the mammal, according to thedisclosure.

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients to a subject so that both activepharmaceutical ingredients and/or their metabolites are present in thesubject at the same time. Co-administration includes simultaneousadministration in separate compositions, administration at differenttimes in separate compositions, or administration in a composition inwhich two or more active pharmaceutical ingredients are present.Simultaneous administration in separate compositions and administrationin a composition in which both agents are present are preferred.

The terms “active pharmaceutical ingredient” and “drug” include the p38αMAPK inhibitors described herein and, more specifically, the p38α MAPKinhibitors described by Formulas A, I, II, 1001-1180, in particularFormulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035(SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087(SF-7-044). The terms “active pharmaceutical ingredient” and “drug” mayalso include those compounds described herein that bind p38α MAPKprotein and thereby modulate p38α MAPK protein activity.

The term “isostere” refers to a group or molecule whose chemical and/orphysical properties are similar to those of another group or molecule. A“bioisostere” is a type of isostere and refers to a group or moleculewhose biological properties are similar to those of another group ormolecule. For example, for the p38α MAPK inhibitors described herein, acarboxylic acid may be replaced by one of the following bioisosteres forcarboxylic acids, including, without limitation, alkyl esters (COOR),acylsulfonamides (CONR—SO₂R), hydroxamic acids (CONR—OH), hydroxamates(CONR—OR), tetrazoles, hydroxyisoxazoles, isoxazol-3-ones, andsulfonamides (SO₂NR), where each R may independently represent hydrogen,alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. In vitro assays encompass cell-based assays in whichcells alive or dead are employed and may also encompass a cell-freeassay in which no intact cells are employed.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound or combination of compounds as describedherein that is sufficient to effect the intended application including,but not limited to, disease treatment. A therapeutically effectiveamount may vary depending upon the intended application (in vitro or invivo), or the subject and disease condition being treated (e.g., theweight, age and gender of the subject), the severity of the diseasecondition, the manner of administration, etc., which can readily bedetermined by one of ordinary skill in the art. The term also applies toa dose that will induce a particular response in target cells (e.g., thereduction of platelet adhesion and/or cell migration). The specific dosewill vary depending on the particular compounds chosen, the dosingregimen to be followed, whether the compound is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichthe compound is carried.

A “therapeutic effect” as that term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit. A prophylactic effectincludes delaying or eliminating the appearance of a disease orcondition, delaying or eliminating the onset of symptoms of a disease orcondition, slowing, halting, or reversing the progression of a diseaseor condition, or any combination thereof.

As used herein, the terms “treat,” “treatment,” and/or “treating” mayrefer to the management of a disease, disorder, or pathologicalcondition, or symptom thereof with the intent to cure, ameliorate,stabilize, and/or control the disease, disorder, pathological conditionor symptom thereof. Regarding control of the disease, disorder, orpathological condition more specifically, “control” may include theabsence of condition progression, as assessed by the response to themethods recited herein, where such response may be complete (e.g.,placing the disease in remission) or partial (e.g., lessening orameliorating any symptoms associated with the condition). As usedherein, the terms “prevent,” “preventing,” and/or “prevention” may referto reducing the risk of developing a disease, disorder, or pathologicalcondition.

As used herein, the terms “modulate” and “modulation” refer to a changein biological activity for a biological molecule (e.g., a protein, gene,peptide, antibody, and the like), where such change may relate to anincrease in biological activity (e.g., increased activity, agonism,activation, expression, upregulation, and/or increased expression) ordecrease in biological activity (e.g., decreased activity, antagonism,suppression, deactivation, downregulation, and/or decreased expression)for the biological molecule. For example, the compounds described hereinmay modulate (i.e., inhibit) p38α MAPK protein. In some embodiments, thecompounds described herein may selectively modulate (i.e., selectivelyinhibit) p38α MAPK protein as compared to other MAPK or p38 MAPKproteins. In some embodiments, the compounds described herein mayselectively modulate (i.e., selectively inhibit) p38α MAPK protein ascompared to other MAPK or p38 MAPK proteins.

The terms “QD,” “qd,” or “q.d.” mean quaque die, once a day, or oncedaily. The terms “BID,” “bid,” or “b.i.d.” mean bis in die, twice a day,or twice daily. The terms “TID,” “tid,” or “t.i.d.” mean ter in die,three times a day, or three times daily. The terms “QID,” “qid,” or“q.i.d.” mean quater in die, four times a day, or four times daily.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions known in the art.Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids. Preferred inorganic acids from whichsalts can be derived include, for example, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid.Preferred organic acids from which salts can be derived include, forexample, acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid andsalicylic acid. Pharmaceutically acceptable base addition salts can beformed with inorganic and organic bases. Inorganic bases from whichsalts can be derived include, for example, sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese andaluminum. Organic bases from which salts can be derived include, forexample, primary, secondary, and tertiary amines, substituted aminesincluding naturally occurring substituted amines, cyclic amines andbasic ion exchange resins. Specific examples include isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. In some embodiments, the pharmaceutically acceptable baseaddition salt is chosen from ammonium, potassium, sodium, calcium, andmagnesium salts. The term “cocrystal” refers to a molecular complexderived from a number of cocrystal formers known in the art. Unlike asalt, a cocrystal typically does not involve hydrogen transfer betweenthe cocrystal and the drug, and instead involves intermolecularinteractions, such as hydrogen bonding, aromatic ring stacking, ordispersive forces, between the cocrystal former and the drug in thecrystal structure.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” or “physiologically compatible” carrier or carrier medium isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and inert ingredients. The use of such pharmaceuticallyacceptable carriers or pharmaceutically acceptable excipients for activepharmaceutical ingredients is well known in the art. Except insofar asany conventional pharmaceutically acceptable carrier or pharmaceuticallyacceptable excipient is incompatible with the active pharmaceuticalingredient, its use in the therapeutic compositions of the invention iscontemplated. Additional active pharmaceutical ingredients, such asother drugs, can also be incorporated into the described compositionsand methods.

A “prodrug” refers to a derivative of a compound described herein, thepharmacologic action of which results from the conversion by chemical ormetabolic processes in vivo to the active compound. Prodrugs includecompounds wherein an amino acid residue, or a polypeptide chain of twoor more (e.g., two, three or four) amino acid residues is covalentlyjoined through an amide or ester bond to a free amino, hydroxyl orcarboxylic acid group of Formulas A, I, II, 1001-1180, in particularFormulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035(SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087(SF-7-044). The amino acid residues include but are not limited to the20 naturally occurring amino acids commonly designated by one or threeletter symbols but also include, for example, 4-hydroxyproline,hydroxylysine, desmosine, isodesmosine, 3-methylhistidine, beta-alanine,gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithineand methionine sulfone. Additional types of prodrugs are alsoencompassed. For instance, free carboxyl groups can be derivatized asamides or alkyl esters (e.g., methyl esters and acetoxy methyl esters).Prodrug esters as employed herein includes esters and carbonates formedby reacting one or more hydroxyls of compounds of the method of theinvention with alkyl, alkoxy, or aryl substituted acylating agentsemploying procedures known to those skilled in the art to generateacetates, pivalates, methylcarbonates, benzoates and the like. Asfurther examples, free hydroxyl groups may be derivatized using groupsincluding but not limited to hemisuccinates, phosphate esters,dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as outlinedin Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs ofhydroxyl and amino groups are also included, as are carbonate prodrugs,sulfonate prodrugs, sulfonate esters and sulfate esters of hydroxylgroups. Free amines can also be derivatized to amides, sulfonamides orphosphonamides. All of the stated prodrug moieties may incorporategroups including but not limited to ether, amine and carboxylic acidfunctionalities. Moreover, any compound that can be converted in vivo toprovide the bioactive agent (e.g., a compound of formula A, I, II, III,and IV) is a prodrug within the scope of the invention. Various forms ofprodrugs are well known in the art. A comprehensive description of prodrugs and prodrug derivatives are described in: (a) The Practice ofMedicinal Chemistry, Camille G. Wermuth et al., (Academic Press, 1996);(b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); (c) ATextbook of Drug Design and Development, P. Krogsgaard-Larson and H.Bundgaard, eds., (Harwood Academic Publishers, 1991). In general,prodrugs may be designed to improve the penetration of a drug acrossbiological membranes in order to obtain improved drug absorption, toprolong duration of action of a drug (slow release of the parent drugfrom a prodrug, decreased first-pass metabolism of the drug), to targetthe drug action (e.g., organ or tumor-targeting, lymphocyte targeting),to modify or improve aqueous solubility of a drug (e.g., i.v.preparations and eyedrops), to improve topical drug delivery (e.g.,dermal and ocular drug delivery), to improve the chemical/enzymaticstability of a drug, or to decrease off-target drug effects, and moregenerally in order to improve the therapeutic efficacy of the compoundsutilized in the invention.

Unless otherwise stated, the chemical structures depicted herein areintended to include compounds which differ only in the presence of oneor more isotopically enriched atoms. For example, compounds where one ormore hydrogen atoms is replaced by deuterium or tritium, or wherein oneor more carbon atoms is replaced by ¹³C- or ¹⁴C-enriched carbons, arewithin the scope of this invention.

When ranges are used herein to describe, for example, physical orchemical properties such as molecular weight or chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. Use of the term “about” whenreferring to a number or a numerical range means that the number ornumerical range referred to is an approximation within experimentalvariability (or within statistical experimental error), and thus thenumber or numerical range may vary. The variation is typically from 0%to 15%, or from 0% to 10%, or from 0% to 5% of the stated number ornumerical range. The term “comprising” (and related terms such as“comprise” or “comprises” or “having” or “including”) includes thoseembodiments such as, for example, an embodiment of any composition ofmatter, method or process that “consist of” or “consist essentially of”the described features.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to ten carbon atoms (e.g., (C₁₋₁₀)alkyl orC₁₋₁₀ alkyl). Whenever it appears herein, a numerical range such as “1to 10” refers to each integer in the given range, e.g., “1 to 10 carbonatoms” means that the alkyl group may consist of 1 carbon atom, 2 carbonatoms, 3 carbon atoms, etc., up to and including 10 carbon atoms,although the definition is also intended to cover the occurrence of theterm “alkyl” where no numerical range is specifically designated.Typical alkyl groups include, but are in no way limited to, methyl,ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl isobutyl,tertiary butyl, pentyl, isopentyl, neopentyl, hexyl, septyl, octyl,nonyl and decyl. The alkyl moiety may be attached to the rest of themolecule by a single bond, such as for example, methyl (Me), ethyl (Et),n-propyl (Pr), 1-methylethyl (isopropyl), n-butyl, n-pentyl,1,1-dimethylethyl (t-butyl) and 3-methylhexyl. Unless stated otherwisespecifically in the specification, an alkyl group is optionallysubstituted by one or more of substituents which are independentlyheteroalkyl, acylsulfonamido, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—S(O)_(t)R^(a)— (where t is 1 or 2), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO(OR^(a))₂ where each R^(a)is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Alkylaryl” refers to an -(alkyl)aryl radical where aryl and alkyl areas disclosed herein and which are optionally substituted by one or moreof the substituents described as suitable substituents for aryl andalkyl respectively.

“Alkylhetaryl” refers to an -(alkyl)hetaryl radical where hetaryl andalkyl are as disclosed herein and which are optionally substituted byone or more of the substituents described as suitable substituents foraryl and alkyl respectively.

“Alkylheterocycloalkyl” refers to an -(alkyl) heterocyclic radical wherealkyl and heterocycloalkyl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for heterocycloalkyl and alkyl respectively.

An “alkene” moiety refers to a group consisting of at least two carbonatoms and at least one carbon-carbon double bond, and an “alkyne” moietyrefers to a group consisting of at least two carbon atoms and at leastone carbon-carbon triple bond. The alkyl moiety, whether saturated orunsaturated, may be branched, straight chain, or cyclic.

“Alkenyl” refers to a straight or branched hydrocarbon chain radicalgroup consisting solely of carbon and hydrogen atoms, containing atleast one double bond, and having from two to ten carbon atoms (i.e.,(C₂₋₁₀)alkenyl or C₂₋₁₀ alkenyl). Whenever it appears herein, anumerical range such as “2 to 10” refers to each integer in the givenrange—e.g., “2 to 10 carbon atoms” means that the alkenyl group mayconsist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10carbon atoms. The alkenyl moiety may be attached to the rest of themolecule by a single bond, such as for example, ethenyl (i.e., vinyl),prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl and penta-1,4-dienyl.Unless stated otherwise specifically in the specification, an alkenylgroup is optionally substituted by one or more substituents which areindependently alkyl, heteroalkyl, acylsulfonamido, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—S(O)_(t)R^(a)— (where t is 1 or 2), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO(OR^(a))₂, where each R^(a)is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Alkenyl-cycloalkyl” refers to an -(alkenyl)cycloalkyl radical wherealkenyl and cycloalkyl are as disclosed herein and which are optionallysubstituted by one or more of the substituents described as suitablesubstituents for alkenyl and cycloalkyl respectively.

“Alkynyl” refers to a straight or branched hydrocarbon chain radicalgroup consisting solely of carbon and hydrogen atoms, containing atleast one triple bond, having from two to ten carbon atoms (i.e.,(C₂₋₁₀)alkynyl or C₂₋₁₀ alkynyl). Whenever it appears herein, anumerical range such as “2 to 10” refers to each integer in the givenrange—e.g., “2 to 10 carbon atoms” means that the alkynyl group mayconsist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10carbon atoms. The alkynyl may be attached to the rest of the molecule bya single bond, for example, ethynyl, propynyl, butynyl, pentynyl andhexynyl. Unless stated otherwise specifically in the specification, analkynyl group is optionally substituted by one or more substituentswhich independently are: alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, hydroxamate, acylsulfonamido, aryl,arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a),—SR^(a), —S(O)_(t)R^(a)— (where t is 1 or 2), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO(OR^(a))₂, where each R^(a)is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Alkynyl-cycloalkyl” refers to an -(alkynyl)cycloalkyl radical wherealkynyl and cycloalkyl are as disclosed herein and which are optionallysubstituted by one or more of the substituents described as suitablesubstituents for alkynyl and cycloalkyl respectively.

“Acylsulfonamide” refers to the group —C(═O)NR^(a)—S(═O)₂R^(a), whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl.

“Carboxaldehyde” refers to a —(C═O)H radical.

“Carbonyl” refers to the group —C(═O)—. Carbonyl groups may besubstituted with the following exemplary substituents: alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,hydroxamate, arylsulfonamido, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—S(O)_(t)R^(a)— (where t is 1 or 2), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —NR^(a)—OR^(a)—, —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)N(R^(a))₂ (where tis 1 or 2), or PO(OR^(a))₂, where each R^(a) is independently hydrogen,alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Carboxyl” refers to a —(C═O)OH radical.

“Cyano” refers to a —CN radical.

“Cycloalkyl” refers to a monocyclic or polycyclic radical that containsonly carbon and hydrogen, and may be saturated, or partiallyunsaturated. Cycloalkyl groups include groups having from 3 to 10 ringatoms (i.e., (C₃₋₁₀)cycloalkyl or C₃₋₁₀ cycloalkyl). Whenever it appearsherein, a numerical range such as “3 to 10” refers to each integer inthe given range—e.g., “3 to 10 carbon atoms” means that the cycloalkylgroup may consist of 3 carbon atoms, etc., up to and including 10 carbonatoms. Illustrative examples of cycloalkyl groups include, but are notlimited to the following moieties: cyclopropyl, cyclobutyl, cyclopentyl,cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl,cyclononyl, cyclodecyl, norbornyl, and the like. Unless stated otherwisespecifically in the specification, a cycloalkyl group is optionallysubstituted by one or more substituents which independently are: alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, acylsulfonamido,heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—S(O)_(t)R^(a)— (where t is 1 or 2), —S(O)_(t)R^(a)— (where t is 1 or2), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)N(R^(a))₂ (where tis 1 or 2), or PO(OR^(a))₂, where each R^(a) is independently hydrogen,alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Cycloalkyl-alkenyl” refers to a -(cycloalkyl)alkenyl radical wherecycloalkyl and alkenyl are as disclosed herein and which are optionallysubstituted by one or more of the substituents described as suitablesubstituents for cycloalkyl and alkenyl, respectively.

“Cycloalkyl-heterocycloalkyl” refers to a -(cycloalkyl)heterocycloalkylradical where cycloalkyl and heterocycloalkyl are as disclosed hereinand which are optionally substituted by one or more of the substituentsdescribed as suitable substituents for cycloalkyl and heterocycloalkyl,respectively.

“Cycloalkyl-heteroaryl” refers to a -(cycloalkyl)heteroaryl radicalwhere cycloalkyl and heteroaryl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for cycloalkyl and heteroaryl, respectively.

The term “alkoxy” refers to the group —O-alkyl, including from 1 to 8carbon atoms of a straight, branched, cyclic configuration andcombinations thereof attached to the parent structure through an oxygen.Examples include, but are not limited to, methoxy, ethoxy, propoxy,isopropoxy, cyclopropyloxy and cyclohexyloxy. “Lower alkoxy” refers toalkoxy groups containing one to six carbons.

The term “substituted alkoxy” refers to alkoxy wherein the alkylconstituent is substituted (i.e., —O-(substituted alkyl)). Unless statedotherwise specifically in the specification, the alkyl moiety of analkoxy group is optionally substituted by one or more substituents whichindependently are: alkyl, heteroalkyl, alkenyl, arylsulfonamido,alkynyl, cycloalkyl, heterocycloalkyl, hydroxamate, aryl, arylalkyl,heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—S(O)_(t)R^(a)— (where t is 1 or 2), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO(OR^(a))₂, where each R^(a)is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

The term “alkoxycarbonyl” refers to a group of the formula(alkoxy)(C═O)— attached through the carbonyl carbon wherein the alkoxygroup has the indicated number of carbon atoms. Thus a(C₁₋₆)alkoxycarbonyl group is an alkoxy group having from 1 to 6 carbonatoms attached through its oxygen to a carbonyl linker. “Loweralkoxycarbonyl” refers to an alkoxycarbonyl group wherein the alkoxygroup is a lower alkoxy group.

The term “substituted alkoxycarbonyl” refers to the group (substitutedalkyl)-O—C(O)— wherein the group is attached to the parent structurethrough the carbonyl functionality. Unless stated otherwise specificallyin the specification, the alkyl moiety of an alkoxycarbonyl group isoptionally substituted by one or more substituents which independentlyare: alkyl, heteroalkyl, arylsulfonamido, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—S(O)_(t)R^(a)— (where t is 1 or 2), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO(OR^(a))₂, where each R^(a)is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Acyl” refers to the groups (alkyl)-C(O)—, (aryl)-C(O)—,(heteroaryl)-C(O)—, (heteroalkyl)-C(O)— and (heterocycloalkyl)-C(O)—,wherein the group is attached to the parent structure through thecarbonyl functionality. If the R radical is heteroaryl orheterocycloalkyl, the hetero ring or chain atoms contribute to the totalnumber of chain or ring atoms. Unless stated otherwise specifically inthe specification, the alkyl, aryl or heteroaryl moiety of the acylgroup is optionally substituted by one or more substituents which areindependently alkyl, heteroalkyl, acylsulfonamido, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—S(O)_(t)R^(a)— (where t is 1 or 2), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO(OR^(a))₂, where each R^(a)is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Acyloxy” refers to a R(C═O)O— radical wherein R is alkyl, aryl,heteroaryl, heteroalkyl or heterocycloalkyl, which are as describedherein. If the R radical is heteroaryl or heterocycloalkyl, the heteroring or chain atoms contribute to the total number of chain or ringatoms. Unless stated otherwise specifically in the specification, the Rof an acyloxy group is optionally substituted by one or moresubstituents which independently are: alkyl, heteroalkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, hydroxamate, aryl, arylalkyl,heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—S(O)_(t)R^(a)— (where t is 1 or 2), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO(OR^(a))₂, where each R^(a)is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Amino” or “amine” refers to a —N(R^(a))₂ radical group, where eachR^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, unless statedotherwise specifically in the specification. When a —N(R^(a))₂ group hastwo R^(a) substituents other than hydrogen, they can be combined withthe nitrogen atom to form a 4-, 5-, 6- or 7-membered ring. For example,—N(R^(a))z is intended to include, but is not limited to, 1-pyrrolidinyland 4-morpholinyl. Unless stated otherwise specifically in thespecification, an amino group is optionally substituted by one or moresubstituents which independently are: alkyl, acylsulfonamido,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,hydroxamate, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,—OR^(a), —SR^(a), —S(O)_(t)R^(a)— (where t is 1 or 2), —OC(O)—R^(a),—N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO(OR^(a))₂, where each R^(a)is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

The term “substituted amino” also refers to N-oxides of the groups—NHR^(d), and NR^(d)R^(d) each as described above. N-oxides can beprepared by treatment of the corresponding amino group with, forexample, hydrogen peroxide or m-chloroperoxybenzoic acid.

“Amide” or “amido” refers to a chemical moiety with formula—C(O)NR^(a)R^(b) or —NR^(a)C(O)R^(b), where R^(a) and R^(b) are selectedfrom the group consisting of hydrogen, alkyl, cycloalkyl, aryl,heteroaryl (bonded through a ring carbon) and heteroalicyclic (bondedthrough a ring carbon), each of which moiety may itself be optionallysubstituted. The R^(a) and R^(b) of —C(O)N R^(a)R^(b) amide mayoptionally be taken together with the nitrogen to which they areattached to form a 4-, 5-, 6- or 7-membered ring. Unless statedotherwise specifically in the specification, an amido group isoptionally substituted independently by one or more of the substituentsas described herein for alkyl, amino, cycloalkyl, aryl, heteroaryl, orheterocycloalkyl. An amide may be an amino acid or a peptide moleculeattached to a compound disclosed herein, thereby forming a prodrug. Theprocedures and specific groups to make such amides are known to those ofskill in the art and can readily be found in seminal sources such asGreene and Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed.,John Wiley & Sons, New York, N.Y., 1999, which is incorporated herein byreference in its entirety.

“Aromatic” or “aryl” or “Ar” refers to an aromatic radical with six toten ring atoms (e.g., C₆-C₁₀ aromatic or C₆-C₁₀ aryl) which has at leastone ring having a conjugated pi electron system which is carbocyclic(e.g., phenyl, fluorenyl, and naphthyl). Bivalent radicals formed fromsubstituted benzene derivatives and having the free valences at ringatoms are named as substituted phenylene radicals. Bivalent radicalsderived from univalent polycyclic hydrocarbon radicals whose names endin “-yl” by removal of one hydrogen atom from the carbon atom with thefree valence are named by adding “-idene” to the name of thecorresponding univalent radical, e.g., a naphthyl group with two pointsof attachment is termed naphthylidene. Whenever it appears herein, anumerical range such as “6 to 10” refers to each integer in the givenrange; e.g., “6 to 10 ring atoms” means that the aryl group may consistof 6 ring atoms, 7 ring atoms, etc., up to and including 10 ring atoms.The term includes monocyclic or fused-ring polycyclic (i.e., rings whichshare adjacent pairs of ring atoms) groups. Unless stated otherwisespecifically in the specification, an aryl moiety is optionallysubstituted by one or more substituents which are independently alkyl,heteroalkyl, acylsulfonamido, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—S(O)_(t)R^(a)— (where t is 1 or 2), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO(OR^(a))₂, where each R^(a)is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Aralkyl” or “arylalkyl” refers to an (aryl)alkyl-radical where aryl andalkyl are as disclosed herein and which are optionally substituted byone or more of the substituents described as suitable substituents foraryl and alkyl respectively.

“Ester” refers to a chemical radical of formula —COOR, where R isselected from the group consisting of alkyl, cycloalkyl, aryl,heteroaryl (bonded through a ring carbon) and heteroalicyclic (bondedthrough a ring carbon). The procedures and specific groups to makeesters are known to those of skill in the art and can readily be foundin seminal sources such as Greene and Wuts, Protective Groups in OrganicSynthesis, 3^(rd) Ed., John Wiley & Sons, New York, N.Y., 1999, which isincorporated herein by reference in its entirety. Unless statedotherwise specifically in the specification, an ester group isoptionally substituted by one or more substituents which independentlyare: alkyl, acylsulfonamido, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—S(O)_(t)R^(a)— (where t is 1 or 2), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO(OR^(a))₂, where each R^(a)is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Fluoroalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more fluoro radicals, as defined above, forexample, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, and the like. The alkyl part of thefluoroalkyl radical may be optionally substituted as defined above foran alkyl group.

“Halo,” “halide,” or, alternatively, “halogen” is intended to meanfluoro, chloro, bromo or iodo. The terms “haloalkyl,” “haloalkenyl,”“haloalkynyl,” and “haloalkoxy” include alkyl, alkenyl, alkynyl andalkoxy structures that are substituted with one or more halo groups orwith combinations thereof. For example, the terms “fluoroalkyl” and“fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, inwhich the halo is fluorine.

“Heteroalkyl,” “heteroalkenyl,” and “heteroalkynyl” refer to optionallysubstituted alkyl, alkenyl and alkynyl radicals and which have one ormore skeletal chain atoms selected from an atom other than carbon, e.g.,oxygen, nitrogen, sulfur, phosphorus or combinations thereof. Anumerical range may be given—e.g., C₁-C₄ heteroalkyl which refers to thechain length in total, which in this example is 4 atoms long. Aheteroalkyl group may be substituted with one or more substituents whichindependently are: alkyl, heteroalkyl, alkenyl, alkynyl,acylsulfonamido, cycloalkyl, heterocycloalkyl, hydroxamate, aryl,arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro,oxo, thioxo, trimethylsilanyl, —OR^(a), —SR^(a), —S(O)_(t)R^(a)— (wheret is 1 or 2), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)N(R^(a))₂ (where tis 1 or 2), or PO(OR^(a))₂, where each R^(a) is independently hydrogen,alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Heteroalkylaryl” refers to an -(heteroalkyl)aryl radical whereheteroalkyl and aryl are as disclosed herein and which are optionallysubstituted by one or more of the substituents described as suitablesubstituents for heteroalkyl and aryl, respectively.

“Heteroalkylheteroaryl” refers to an -(heteroalkyl)heteroaryl radicalwhere heteroalkyl and heteroaryl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for heteroalkyl and heteroaryl, respectively.

“Heteroalkylheterocycloalkyl” refers to an-(heteroalkyl)heterocycloalkyl radical where heteroalkyl andheterocycloalkyl are as disclosed herein and which are optionallysubstituted by one or more of the substituents described as suitablesubstituents for heteroalkyl and heterocycloalkyl, respectively.

“Heteroalkylcycloalkyl” refers to an -(heteroalkyl)cycloalkyl radicalwhere heteroalkyl and cycloalkyl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for heteroalkyl and cycloalkyl, respectively.

“Heteroaryl” or “heteroaromatic” or “HetAr” refers to a 5- to18-membered aromatic radical (e.g., C₅-C₁₃ heteroaryl) that includes oneor more ring heteroatoms selected from nitrogen, oxygen and sulfur, andwhich may be a monocyclic, bicyclic, tricyclic or tetracyclic ringsystem. Whenever it appears herein, a numerical range such as “5 to 18”refers to each integer in the given range—e.g., “5 to 18 ring atoms”means that the heteroaryl group may consist of 5 ring atoms, 6 ringatoms, etc., up to and including 18 ring atoms. Bivalent radicalsderived from univalent heteroaryl radicals whose names end in “-yl” byremoval of one hydrogen atom from the atom with the free valence arenamed by adding “-idene” to the name of the corresponding univalentradical—e.g., a pyridyl group with two points of attachment is apyridylidene. A N-containing “heteroaromatic” or “heteroaryl” moietyrefers to an aromatic group in which at least one of the skeletal atomsof the ring is a nitrogen atom. The polycyclic heteroaryl group may befused or non-fused. The heteroatom(s) in the heteroaryl radical areoptionally oxidized. One or more nitrogen atoms, if present, areoptionally quaternized. The heteroaryl may be attached to the rest ofthe molecule through any atom of the ring(s). Examples of heteroarylsinclude, but are not limited to, azepinyl, acridinyl, benzimidazolyl,benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl,benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl,benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzofurazanyl,benzothiazolyl, benzothienyl(benzothiophenyl),benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,cyclopenta[d]pyrimidinyl,6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl,5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl,6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl,dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo[3,2-c]pyridinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl,indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl,isoquinolyl, indolizinyl, isoxazolyl, isoxazol-3-one,5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl,5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl,pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl,pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl,quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl,5,6,7,8-tetrahydroquinazolinyl,5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl,5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl,thiapyranyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl,thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl (i.e.thienyl). Unless stated otherwise specifically in the specification, aheteroaryl moiety is optionally substituted by one or more substituentswhich are independently: alkyl, acylsulfonamido, heteroalkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, hydroxamate, aryl, arylalkyl,heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo,trimethylsilanyl, —OR^(a), —SR^(a), —S(O)_(t)R^(a)— (where t is 1 or 2),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)N(R^(a))₂ (where tis 1 or 2), or PO(OR^(a))₂, where each R^(a) is independently hydrogen,alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

Substituted heteroaryl also includes ring systems substituted with oneor more oxide (—O—) substituents, such as, for example, pyridinylN-oxides.

“Heteroarylalkyl” refers to a moiety having an aryl moiety, as describedherein, connected to an alkylene moiety, as described herein, whereinthe connection to the remainder of the molecule is through the alkylenegroup.

“Heterocycloalkyl” refers to a stable 3- to 18-membered non-aromaticring radical that comprises two to twelve carbon atoms and from one tosix heteroatoms selected from nitrogen, oxygen and sulfur. Whenever itappears herein, a numerical range such as “3 to 18” refers to eachinteger in the given range—e.g., “3 to 18 ring atoms” means that theheterocycloalkyl group may consist of 3 ring atoms, 4 ring atoms, etc.,up to and including 18 ring atoms. Unless stated otherwise specificallyin the specification, the heterocycloalkyl radical is a monocyclic,bicyclic, tricyclic or tetracyclic ring system, which may include fusedor bridged ring systems. The heteroatoms in the heterocycloalkyl radicalmay be optionally oxidized. One or more nitrogen atoms, if present, areoptionally quaternized. The heterocycloalkyl radical is partially orfully saturated. The heterocycloalkyl may be attached to the rest of themolecule through any atom of the ring(s). Examples of suchheterocycloalkyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in thespecification, a heterocycloalkyl moiety is optionally substituted byone or more substituents which independently are: alkyl,acylsulfonamido, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo,trimethylsilanyl, —OR^(a), —SR^(a), —S(O)_(t)R^(a)— (where t is 1 or 2),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)N(R^(a))₂ (where tis 1 or 2), or PO(OR^(a))₂, where each R^(a) is independently hydrogen,alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Heterocycloalkyl” also includes bicyclic ring systems wherein onenon-aromatic ring, usually with 3 to 7 ring atoms, contains at least 2carbon atoms in addition to 1-3 heteroatoms independently selected fromoxygen, sulfur, and nitrogen, as well as combinations comprising atleast one of the foregoing heteroatoms; and the other ring, usually with3 to 7 ring atoms, optionally contains 1-3 heteroatoms independentlyselected from oxygen, sulfur, and nitrogen and is not aromatic.

“Hydroxamate” refers to the —C(O)NR^(a)OR^(a) moiety, where each R^(a)is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Nitro” refers to the —NO₂ radical.

“Oxa” refers to the —O— radical.

“Oxo” refers to the ═O radical.

“Isomers” are different compounds that have the same molecular formula.“Stereoisomers” are isomers that differ only in the way the atoms arearranged in space—i.e., having a different stereochemical configuration.“Enantiomers” are a pair of stereoisomers that are non-superimposablemirror images of each other. A 1:1 mixture of a pair of enantiomers is a“racemic” mixture. The term “(±)” is used to designate a racemic mixturewhere appropriate. “Diastereoisomers” are stereoisomers that have atleast two asymmetric atoms, but which are not mirror-images of eachother. The absolute stereochemistry is specified according to theCahn-Ingold-Prelog R—S system. When a compound is a pure enantiomer thestereochemistry at each chiral carbon can be specified by either (R) or(S). Resolved compounds whose absolute configuration is unknown can bedesignated (+) or (−) depending on the direction (dextro- orlevorotatory) which they rotate plane polarized light at the wavelengthof the sodium D line. Certain of the compounds described herein containone or more asymmetric centers and can thus give rise to enantiomers,diastereomers, and other stereoisomeric forms that can be defined, interms of absolute stereochemistry, as (R) or (S). The present chemicalentities, pharmaceutical compositions and methods are meant to includeall such possible isomers, including racemic mixtures, optically pureforms and intermediate mixtures. Optically active (R)- and (S)-isomerscan be prepared using chiral synthons or chiral reagents, or resolvedusing conventional techniques. When the compounds described hereincontain olefinic double bonds or other centers of geometric asymmetry,and unless specified otherwise, it is intended that the compoundsinclude both E and Z geometric isomers.

“Enantiomeric purity” as used herein refers to the relative amounts,expressed as a percentage, of the presence of a specific enantiomerrelative to the other enantiomer. For example, if a compound, which maypotentially have an (R)- or an (S)-isomeric configuration, is present asa racemic mixture, the enantiomeric purity is about 50% with respect toeither the (R)- or (S)-isomer. If that compound has one isomeric formpredominant over the other, for example, 80% (S)-isomer and 20%(R)-isomer, the enantiomeric purity of the compound with respect to the(S)-isomeric form is 80%. The enantiomeric purity of a compound can bedetermined in a number of ways known in the art, including but notlimited to chromatography using a chiral support, polarimetricmeasurement of the rotation of polarized light, nuclear magneticresonance spectroscopy using chiral shift reagents which include but arenot limited to lanthanide containing chiral complexes or Pirkle'sreagents, or derivatization of a compounds using a chiral compound suchas Mosher's acid followed by chromatography or nuclear magneticresonance spectroscopy.

In some embodiments, the enantiomerically enriched composition has ahigher potency with respect to therapeutic utility per unit mass thandoes the racemic mixture of that composition. Enantiomers can beisolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred enantiomerscan be prepared by asymmetric syntheses. See, for example, Jacques, etal., Enantiomers, Racemates and Resolutions, Wiley Interscience, NewYork (1981); E. L. Eliel, Stereochemistry of Carbon Compounds,McGraw-Hill, New York (1962); and E. L. Eliel and S. H. Wilen,Stereochemistry of Organic Compounds, Wiley-Interscience, New York(1994).

The terms “enantiomerically enriched” and “non-racemic,” as used herein,refer to compositions in which the percent by weight of one enantiomeris greater than the amount of that one enantiomer in a control mixtureof the racemic composition (e.g., greater than 1:1 by weight). Forexample, an enantiomerically enriched preparation of the (S)-enantiomer,means a preparation of the compound having greater than 50% by weight ofthe (S)-enantiomer relative to the (R)-enantiomer, such as at least 75%by weight, or such as at least 80% by weight. In some embodiments, theenrichment can be significantly greater than 80% by weight, providing a“substantially enantiomerically enriched” or a “substantiallynon-racemic” preparation, which refers to preparations of compositionswhich have at least 85% by weight of one enantiomer relative to otherenantiomer, such as at least 90% by weight, or such as at least 95% byweight. The terms “enantiomerically pure” or “substantiallyenantiomerically pure” refers to a composition that comprises at least98% of a single enantiomer and less than 2% of the opposite enantiomer.

“Moiety” refers to a specific segment or functional group of a molecule.Chemical moieties are often recognized chemical entities embedded in orappended to a molecule.

“Tautomers” are structurally distinct isomers that interconvert bytautomerization. “Tautomerization” is a form of isomerization andincludes prototropic or proton-shift tautomerization, which isconsidered a subset of acid-base chemistry. “Prototropictautomerization” or “proton-shift tautomerization” involves themigration of a proton accompanied by changes in bond order, often theinterchange of a single bond with an adjacent double bond. Wheretautomerization is possible (e.g., in solution), a chemical equilibriumof tautomers can be reached. An example of tautomerization is keto-enoltautomerization. A specific example of keto-enol tautomerization is theinterconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-onetautomers. Another example of tautomerization is phenol-ketotautomerization. A specific example of phenol-keto tautomerization isthe interconversion of pyridin-4-ol and pyridin-4(1H)-one tautomers.

A “leaving group or atom” is any group or atom that will, under selectedreaction conditions, cleave from the starting material, thus promotingreaction at a specified site. Examples of such groups, unless otherwisespecified, include halogen atoms and mesyloxy, p-nitrobenzensulphonyloxyand tosyloxy groups.

“Protecting group” is intended to mean a group that selectively blocksone or more reactive sites in a multifunctional compound such that achemical reaction can be carried out selectively on another unprotectedreactive site and the group can then be readily removed or deprotectedafter the selective reaction is complete. A variety of protecting groupsare disclosed, for example, in T. H. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons,New York (1999).

“Solvate” refers to a compound in physical association with one or moremolecules of a pharmaceutically acceptable solvent.

“Substituted” means that the referenced group may have attached one ormore additional groups, radicals or moieties individually andindependently selected from, for example, acyl, alkyl, alkylaryl,cycloalkyl, aralkyl, aryl, carbohydrate, carbonate, heteroaryl,heterocycloalkyl, hydroxamate, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, cyano, halo, carbonyl, ester, thiocarbonyl,isocyanato, thiocyanato, isothiocyanato, nitro, oxo, perhaloalkyl,perfluoroalkyl, phosphate, silyl, sulfinyl, sulfonyl, sulfonamidyl,sulfoxyl, sulfonate, urea, and amino, including mono- and di-substitutedamino groups, and protected derivatives thereof. The substituentsthemselves may be substituted, for example, a cycloalkyl substituent mayitself have a halide substituent at one or more of its ring carbons. Theterm “optionally substituted” means optional substitution with thespecified groups, radicals or moieties.

“Sulfanyl” refers to groups that include —S-(optionally substitutedalkyl), —S-(optionally substituted aryl), —S-(optionally substitutedheteroaryl) and —S-(optionally substituted heterocycloalkyl).

“Sulfinyl” refers to groups that include —S(O)—H, —S(O)-(optionallysubstituted alkyl), —S(O)-(optionally substituted amino),—S(O)-(optionally substituted aryl), —S(O)-(optionally substitutedheteroaryl) and —S(O)-(optionally substituted heterocycloalkyl).

“Sulfonyl” refers to groups that include —S(O₂)—H, —S(O₂)-(optionallysubstituted alkyl), —S(O₂)-(optionally substituted amino),—S(O₂)-(optionally substituted aryl), —S(O₂)-(optionally substitutedheteroaryl), and —S(O₂)-(optionally substituted heterocycloalkyl).

“Sulfonamidyl” or “sulfonamido” refers to a —S(═O)₂—NRR radical, whereeach R is selected independently from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andheteroalicyclic (bonded through a ring carbon). The R groups in —NRR ofthe —S(═O)₂—NRR radical may be taken together with the nitrogen to whichit is attached to form a 4-, 5-, 6- or 7-membered ring. A sulfonamidogroup is optionally substituted by one or more of the substituentsdescribed for alkyl, cycloalkyl, aryl, heteroaryl, respectively.

“Sulfoxyl” refers to a —S(═O)₂OH radical.

“Sulfonate” refers to a —S(═O)₂—OR radical, where R is selected from thegroup consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded througha ring carbon) and heteroalicyclic (bonded through a ring carbon). Asulfonate group is optionally substituted on R by one or more of thesubstituents described for alkyl, cycloalkyl, aryl, heteroaryl,respectively.

Compounds of the invention also include crystalline and amorphous formsof those compounds, including, for example, polymorphs,pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (includinganhydrates), conformational polymorphs, and amorphous forms of thecompounds, as well as mixtures thereof. “Crystalline form” and“polymorph” are intended to include all crystalline and amorphous formsof the compound, including, for example, polymorphs, pseudopolymorphs,solvates, hydrates, unsolvated polymorphs (including anhydrates),conformational polymorphs, and amorphous forms, as well as mixturesthereof, unless a particular crystalline or amorphous form is referredto.

p38 Mitogen-Activated Protein Kinases (MAPKs), Inhibition Thereof, andp38α Selective Inhibition

The p38 mitogen-activated protein kinase (MAPKs) family of stress- andcytokine-activated kinases contribute to the pathogenesis of many humandiseases, including cancer, rheumatoid arthritis, cardiovasculardisease, multiple sclerosis, inflammatory bowel disease, chronicobstructive pulmonary disease (COPD), asthma, acute respiratory distresssyndrome (ARDS), and acute lung injury (ALI). Among the many importantbiological processes regulated by p38 MAPKs, regulation of endothelialand epithelial barrier function, leukocyte trafficking, and cytokineexpression are central to the pathogenesis of acute and chronicinflammatory disorders. While preclinical studies support pharmacologicinhibition of p38 as promising treatment for inflammatory diseases, p38inhibitors have had very limited success in clinical testing because ofdose-limiting toxicity and lack of efficacy. Of the 36 Phase II clinicaltrials of p38 inhibitors listed in www.clinicaltrials.gov, results ofonly 8 studies have been published or listed in ClinicalTrials.gov andshowed little clinical benefit and moderate toxicity.

All available p38 inhibitors block catalytic activity either by directlycompeting for ATP binding or by allosterically causing conformationalchanges that preclude access of ATP to the catalytic site. Davidson etal. identified a purported p38α substrate-selective inhibitor, CMPD1,which selectively inhibited MK2 phosphorylation in in vitro kinaseassays, but it bound near the p38α active site and was subsequentlyshown to lack substrate-selectivity in cells. Almost all availableinhibitors are active against both p38α and p38ß, and some are activeagainst additional isoforms. Yet genetic and pharmacologic studies haveidentified p38α as the proinflammatory isoform, while other studies havedemonstrated p38β signaling to be cytoprotective. Therefore, inhibitionof p38β may contribute to both lack of efficacy and toxicity ofnon-isoform-selective p38 inhibitors. However, the extensive structuralconservation of the catalytic module across most protein kinasespresents a challenge to developing catalytic inhibitors with highselectivity, especially for individual p38 isoforms.

Without wishing to be bound by any particular theory, it is believedthat even if the catalytic inhibitors were absolutely selective forp38α, these compounds by design would block all p38α signaling events,many of which are essential for reestablishing and maintaininghomeostasis. For example, p38α not only activates expression ofproinflammatory cytokines, it also activates anti-inflammatory cytokinesand counterregulatory dual specificity protein phosphatase-2 (DUSP2)through the p38α substrate, MSK1/2. The transient decrease andsubsequent rebound of serum C-reactive protein (CRP) levels seen inclinical trials of p38 catalytic inhibitors might be caused by loss ofthe MSK1/2-dependent anti-inflammatory signaling.

As an alternative to the catalytic inhibitors, in some embodiments, thecompounds and methods of the invention target the substrate bindinggroove of p38α, which stretches between two acidic patches, the CD andED domains, and is distinct from the DEF substrate-binding pocket.Downstream substrates, upstream activating kinases, and possiblyscaffolding molecules, all interact with p38 through these sites. Insome embodiments, computer-aided drug design (CADD) and/or rationaldesign were used in order to target low molecular weight compounds to apocket near the p38α ED substrate binding site, which is required forphosphorylation of MAPK-activated protein kinase-2 (MAPKAPK2; MK2), ap38α substrate known to mediate endothelial permeability and neutrophiltransendothelial migration (TEM) in vitro, and pulmonary edema in amouse lung injury model; whereas anti-inflammatory MSK1/2 binds to theCD site. Using this algorithm, p38α-binding compounds with highefficiency were identified, compounds able to: stabilize endothelialbarrier function in human lung microvascular endothelial cells(HMVECLs), inhibit LPS-induced proinflammatory gene expression in THP1cells, and be well tolerated and more potent than SB203580 in mitigatingexperimental ALI.

Acute Lung Injury Treated by the p38α MAPK Inhibitors and MethodsDescribed Herein

Acute respiratory distress syndrome (ARDS) is a common cause ofrespiratory failure, which has 30-40% mortality and no effectivetherapeutic options. p38 signaling is important in ARDS pathogenesis,but clinical trials of p38 inhibitors, all of which inactivate the p38catalytic site and block phosphorylation of all p38 substrates, havebeen disappointing due to dose-limiting toxicity.

Acute respiratory distress syndrome (ARDS) is characterized by acuteonset of non-hydrostatic pulmonary edema caused predominantly byneutrophil-mediated injury to the alveolar epithelium and capillaryendothelial barrier dysfunction. A complex network of pro- andanti-inflammatory mediators activated in association with ARDS iscritical to the pathogenesis of acute lung injury (ALI) as well as themultiple organ failure that often accompanies ARDS. However, therapeuticagents that target proinflammatory mediators have proven to beineffective in ARDS. Injury to lung parenchyma causes reducedcompliance, intrapulmonary shunting, and mismatchedventilation-perfusion that usually necessitates mechanical ventilation.However, the cyclical recruitment/de-recruitment of alveoli andoverdistension caused by mechanical ventilation can itself causeneutrophil-dependent inflammation and lung injury even to previouslynormal lungs. Appreciation of this mechanism led to a Phase IIIrandomized clinical trial demonstrating that mechanical ventilation withlow tidal volumes improves survival in patients with ARDS. Twoadditional supportive maneuvers have been shown to improve mortality inpatients with severe ARDS, neuromuscular blockade and prone positioning.A third intervention, conservative fluid management, was shown todecrease duration of mechanical ventilation and ICU length of stay, butnot mortality. Despite these improvements in supportive care, mortalityin patients with ARDS remains 30-40% with about 74,500 deaths per yearin the United States, underscoring the importance of developing newtherapeutics that target the relevant pathogenic mechanisms.

The p38 mitogen-activated protein kinases (MAPKs) are a family ofstress- and cytokine-activated kinases that are activated by many of thepathogenic signals associated with ARDS, including inflammatorymediators, febrile-range hyperthermia (FRH), and cyclic stretch. Sincep38 MAPK is activated in patients at-risk for ARDS and, as discussedbelow, p38 MAPK participates in multiple processes that contribute tothe pathogenesis of ARDS, this family of MAPKs presents an intriguingtherapeutic target in ARDS. As proof of this concept, the prototypicalpyridinyl imidazole compound SB203580, which inhibits kinase activity ofp38α and β, but not p38γ or δ has been shown to block multiple processesthat contribute to the pathogenesis of ARDS. Endothelial p38 signalingis activated by neutrophil binding and required for neutrophiltransendothelial migration (TEM). Cross-linking ICAM-1 on humanumbilical vein endothelial cells (HUVECs) stimulates p38α activation,HSP27 phosphorylation, F-actin rearrangement, ICAM-1 aggregation, andcell stiffening, and increases migration of neutrophils to HUVECintercellular junctions, all of which is blocked by SB203580.Cross-linking E-selectin on HUVECs activates p38 and p38-dependentcytoskeleton rearrangement, stress fiber formation and neutrophil TEM.Similarly, ICAM-1 ligation of ß82 integrins on neutrophils activatesneutrophil p38 and p38-dependent chemokinesis and chemotaxis.Pretreatment with p38 inhibitor was protective in a mouse model ofventilator-induced lung injury, complement-induced lung injury, and lunginjury associated with a cecal-ligation and puncture model of sepsis,but not hemorrhage and endotoxemia-induced lung injury. It was shownthat the exaggerated endothelial barrier dysfunction caused by FRH (2-3°C. increase in core temperature) in experimental ALI is associated withp38 activation and blocked by SB203580. The effectiveness of SB203580 inmitigating multiple pathogenic processes that contribute to acute lunginjury and the relatively high expression of p38α and β in human lungsupports a central role for these two p38 isoforms in the pathogenesisof ARDS.

Despite these persuasive preclinical data there has been only oneclinical trial that begins to evaluate p38 inhibition as a therapeuticstrategy in ARDS. This early phase IIa trial of SB-681323/dilmapimod inpatients at-risk for ARDS (clinicaltrials.gov no. NCT00996840) showeddilmapimod was safe at the doses administered and modestly reduced serumC-reactive protein (CRP) levels, but was not powered to analyze effectson ARDS incidence or severity. There are currently a total of 74clinical trials of p38 inhibitors listed in www.clinicaltrials.gov,including 26 Phase I, 47 Phase II, and one Phase III trial. Phase II andIII trials tested safety and efficacy of ten different p38 catalyticinhibitors for 13 different disease/indications including analgesia (6trials), osteoarthritis (2 trials), rheumatoid arthritis (13 trials),Alzheimer's disease (2 trials), ankylosing spondylitis (1 trial),cardiomyopathy (1 trial), psoriasis (2 trials), atherosclerosis (5trials), depression (2 trials), COPD (8 trials), at-risk ARDS (1 trial),cancer (4 trials), and glomerulosclerosis (1 trial). Although only aportion of the data is in the public domain, the failure of most ofthese drugs seems to have been due to adverse side-effect profiles orlack of effectiveness at the doses used. Of the 48 Phase II and IIItrials 36 have been completed and 3 terminated early, but results ofonly 8 studies have been published or listed in ClinicalTrials.gov. Twotrials of VX-702 in rheumatoid arthritis showed small increases inproportion of treated subjects with ACR20 symptom score vs. placebo. Oftwo published studies of p38 inhibitors for pain, one reported modestreduction in pain and the other no effect. Of two published studies ofp38 inhibitors in COPD, one showed no effect and the other showed a 100ml increase in FEV1 and a decrease in serum CRP levels in the treatmentgroup, but with associated toxicity (rash, pharyngitis, prolonged QTc).GW85655 (losmapimod), improved vascular relaxation and reduced serum CRPin patients with hypercholesterolemia. In a ninth clinical trial, whichwas not listed in clinicaltrials.gov, BIRB 796 (doramapimod) had noclinical effect in patients with Crohn's disease but transiently reducedserum CRP levels. Collectively, these studies demonstrate thetherapeutic potential of p38 inhibition in a broad range of humandisease, but underscore the limited efficacy of the currently availablep38 inhibitors at doses that can be safely administered to humans.

The p38 MAPKs, like most protein kinases, share a conserved bi-lobedstructure and a catalytic site, with its hydrophobic ATP-binding pocket,located between the N-terminal and C-terminal lobes. Most availableprotein kinase inhibitors compete with ATP for binding to theATP-binding pocket of the catalytic site, but the extensive structuralconservation of the catalytic module across most protein kinasespresents a challenge to developing catalytic p38 inhibitors with highspecificity. Since the pyridinyl imidazole inhibitor SB203580 binds theATP-binding site of p38α and β, but its access to the ATP binding siteof p38γ and δ is blocked by a bulky methionine, it is used as a specificinhibitor of p38α and β. However, proteomic analysis identified severaladditional kinases that were inhibited by SB203580 with sub-micromolarIC₅₀, including Rip-like interacting caspase-like apoptosis-regulatoryprotein kinase (RICK/Rip2), casein kinase (CK)-1δ, and cyclinG-associated kinase (GAK).

A new class of diaryl urea compounds was discovered in a high throughputbiochemical screen for p38 inhibitors. Rather than bind directly to theATP binding pocket, these compounds bind to an allosteric site thatinduces a conformational change in p38 that precludes access of ATP toits binding pocket in the catalytic site. Three allosteric p38inhibitors, BIRB 796/dormapimod, GW856553/losmapimod, andSB-681323/dilmapimod have entered clinical testing, but like theATP-competitive, have not progressed beyond phase II testing except forthe LATITUDE study, an ongoing Phase III trial of losmapimod in patientswith acute coronary syndrome (clinicaltrials.gov no. NCT02145468). Sincethe allosteric inhibitors are not affected by the presence of thegatekeeper methionine, these compounds inhibit all four p38 isoforms,but BIRB 796 also potently inhibits Jnk2α2 with IC₅₀ of 0.1 μM andc-Raf-1 with IC₅₀ 1.4 μM. The lack of specificity of the ATP-competitiveand allosteric p38 inhibitors is likely a major source of off-targettoxicity.

An equally important source of p38 inhibitor toxicity likely derivesfrom the broad range of functions of each p38 MAPK isoform. Since bothtypes of inhibitors block the p38 catalytic site, the ATP-competitiveand allosteric inhibitors block all p38 phosphorylation events. Sincep38 phosphorylates at least 66 recognized substrates with importantbiological activity, dose-limiting toxicity may be unavoidable withthese agents.

MAPK p38 and ERK family members share a structural feature, a substratebinding groove located on the C-terminal lobe of the protein on the sideopposite the catalytic domain. The binding groove stretches between twoacidic patches, the CD and ED domains. This region of p38 not only bindsp38 substrates but also binds upstream kinases and scaffolding proteins.Our group has previously developed a new class of ERK1/2 MAPK inhibitorswith improved toxicity profile by using computer-aided drug design(CADD) to identify small molecules that target the substrate bindinggroove rather than the catalytic module of ERK2. As described herein, asimilar strategy may be employed to identify low molecular weightcompounds targeting a pocket near the p38α ED substrate binding site,which is required for phosphorylation of MK2, a p38 substrate known tomediate pulmonary endothelial permeability in vitro and pulmonary edemain a mouse lung injury model. Using CADD to search a database ofcommercially available compounds, 150 low molecular weight compoundspredicted to bind to the targeted pocket near the ED binding site ofp38α target have been identified. Twenty structurally distinct compoundsfrom this list were obtained, screened for selective binding to p38α butnot ERK2 by differential scanning fluorimetry (DSF), then analyzed forcapacity to reduce pathogenic endothelial barrier changes in human lungmicrovascular endothelial cells (HMVECLs) and cytokine expression inTHP1 monocytes in vitro, and to mitigate ALI induced in mice. Of the 20CADD-selected compounds tested, five bound to p38α with sufficientaffinity to detect by DSF, two bound selectively to p38α but not ERK2and were more effective than SB203580 in stabilizing endothelial barrierfunction in vitro, and one of these compounds was well tolerated andmore potent than SB203580 in mitigating experimental ALI.

In certain embodiments, the p38α MAPK inhibitors described herein may beused in the treatment of acute respiratory distress syndrome (ARDS)and/or acute lung injury (ALI).

p38α MAPK Inhibitors and Methods of Inhibiting p38α MAPK

In an embodiment, the invention includes compounds that may be p38α MAPKinhibitors and/or modulators of p38α MAPK protein activity, for examplecompounds capable of binding to a pocket near the ED substrate-dockingsite of p38α MAPK, or a pharmaceutically acceptable salt, solvate,hydrate, cocrystal, or prodrug thereof. In one embodiment, the p38α MAPKinhibitor is a p38α MAPK selective inhibitor. In an embodiment, the p38αMAPK inhibitor binds p38α MAPK near the substrate binding groove of p38αMAPK, which stretches between two acidic patches, the CD and ED domains.In other embodiments, the p38α MAPK inhibitor causes inhibition of MK2phosphorylation.

In one embodiment, a lead p38α MAPK inhibitor compound has beenidentified, that has favorable biological effects in human cell culturemodels and in a mouse model of inflammatory lung injury. In oneembodiment, a p38α MAPK inhibitor has been identified by means of a CADDstrategy. The CADD-targeted pocket in p38α differed from thecorresponding pocket in p38β in 3 of 10 amino acids, which provided anopportunity for p38α-selectivity. In some embodiments, the sequence ofthe targeted pocket at least includes amino acids R49, H107, L108, andK165 in p38α MAPK. In some embodiments, the sequence of the targetedpocket is R49, H107, L108, M109, G110, A157, V158, E163, L164, and K165in p38α MAPK. In one embodiment, a p38α MAPK inhibitor has beenidentified by means of a rational design strategy.

In some embodiments, the p38α MAPK inhibitor is a compound is a compoundof Formula A, or a pharmaceutically acceptable salt, solvate, hydrate,cocrystal, or prodrug thereof:

wherein in Formula A: Ar and Ar¹ are independently selected from a mono-or polycyclic optionally substituted cycloalkyl, mono- or polycyclicoptionally substituted heterocycloalkyl, mono- or polycyclic optionallysubstituted aryl, mono- or polycyclic optionally substituted arylalkyl,mono- or polycyclic optionally substituted heteroaryl, and mono- orpolycyclic optionally substituted heteroarylalkyl; R¹ and R² areindependently selected from hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substituted alkylheteroaryl,wherein R¹ and R² can optionally be joined to form a carbo- orheterocycle; L¹ and L² are linkers comprising independently one or moreof a bond, —NR^(a)—, —S—, —S(O)—, —S(O)₂—, —O—, —CR^(a)2-, —C(O)O—,—OC(O)—, —C(O)S—, —SC(O)—, —C(O)NR^(a)—, —NR^(a)C(O)—, —C(O)NR^(a)SO₂—,—SO₂NR^(a) C(O)—, —OC(O)O—, —OC(O)S—, —SC(O)O—, —OC(O)NR^(a)—,—NR^(a)C(O)O—, —S(O)_(t)N(R^(a))— (where t is 1 or 2),—N(R^(a))S(O)_(t)— (where t is 1 or 2), disubstituted alkyl,disubstituted heteroalkyl, disubstituted alkenyl, disubstituted alkynyl,disubstituted cycloalkyl, disubstituted heterocycloalkyl, disubstitutedaryl, disubstituted arylalkyl, disubstituted heteroaryl, anddisubstituted heteroarylalkyl; wherein any optional substituent isindependently selected at each occurrence from optionally substitutedalkyl, optionally substituted heteroalkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionallysubstituted aryl, optionally substituted arylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —SC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a),—C(O)OR^(a), —C(O)SR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,—N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2),—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), and PO₃(R^(a))₂; and R^(a) isindependently selected at each occurrence from hydrogen, optionallysubstituted alkyl, optionally substituted fluoroalkyl, optionallysubstituted carbocyclyl, optionally substituted carbocyclylalkyl,optionally substituted aryl, optionally substituted aralkyl, optionallysubstituted heterocycloalkyl, optionally substitutedheterocycloalkylalkyl, optionally substituted heteroaryl, and optionallysubstituted heteroarylalkyl. In some embodiments, an optionalsubstituent is independently selected from R³, R⁴, R⁵, and R⁶, which areindependently selected from hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substituted alkylheteroaryl,wherein any two of R¹, R², R³, R⁴, R⁵, and R⁶ can optionally be joinedto form a carbo- or heterocycle. In some embodiments, Ar¹ is anoptionally substituted aryl or optionally substituted heteroaryl ring.In some embodiments, Ar is a mono- or polycyclic optionally substitutedaryl, a mono- or polycyclic optionally substituted arylalkyl, a mono- orpolycyclic optionally substituted heteroaryl, or a mono- or polycyclicoptionally substituted heteroarylalkyl. In some embodiments, Ar is a 5or 6 membered optionally substituted aryl or a 5 or 6 memberedoptionally substituted heteroaryl, wherein the L¹ and L² are connectedto Ar in a 1,2, 1,3, or 1,4 substitution pattern. In some embodiments,Ar is 1,2 disubstituted, 1,3 disubstituted, or 1,4 disubstituted phenyl,pyridine, pyrimidine, pyrazine, or triazine. In some embodiments, Ar is1,2 disubstituted or 1,3 disubstituted furan, thiophene, pyrrole,thiazole, imidazole, oxazole, triazole, or pyrazole. In someembodiments, Ar is 1,2 disubstituted, 1,3 disubstituted, 1,4disubstituted, 1,5 disubstituted, 1,6 disubstituted, 1,7 disubstituted,or 1,8 disubstituted naphthalene, quinoline, isoquinoline, orquinazoline. In some embodiments, Ar is 1,2 disubstituted, 1,3disubstituted, 1,4 disubstituted, 1,5 disubstituted, 1,6 disubstituted,or 1,7 disubstituted indole or imidazole. In some embodiments, L¹ is alinker selected from —CH₂—, —C(CH₃)₂—, and —C(CH₂CH₂)—; L² is a linkerselected from —NHCH₂—, —CH₂NH—, —NHCO—, —CONH—, —SO₂NH—, and —NHSO₂—;and —NR¹R² is selected from:

wherein R⁷ is hydrogen or an optionally substituted alkyl, and Ar² is aheterocycle.

In one embodiment, the p38 MAPK inhibitor is a compound of Formula I, ora pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof:

wherein in Formula L: each of R¹, R², R³, R⁴, R⁵, and R⁶ isindependently selected from hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substituted alkylheteroaryl,wherein R¹ and R² can optionally be joined to form a carbo- orheterocycle; L¹ is a linker selected from —CH₂—, —C(CH₃)₂—, and—C(CH₂CH₂)—; L² is a linker selected from —NHCH₂—, —CH₂NH—, —NHCO—,—CONH—, —SO₂NH—, and —NHSO₂—; and Ar¹ is an optionally substituted arylor optionally substituted heteroaryl ring. In some embodiments, the—NR¹R² group in Formula I is selected from:

wherein R⁷ is hydrogen or an optionally substituted alkyl, and Ar² is aheterocycle. In some embodiments, the —NR¹R² group in Formula I isselected from:

In some embodiments, L¹ is —CH₂—. In some embodiments, R³, R⁴, R⁵, andR⁶ are hydrogens. In some embodiments, L² is selected from —NHCH₂—,—NHCO—, and —NHSO₂—. In some embodiments, Ar¹ is selected from:

wherein each of R⁷, R⁸, R⁹, R¹⁰, R, R¹², and R¹³ is independentlyselected from hydrogen, halogen, —NR¹R², alkoxy, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substituted alkylheteroaryl, andwherein any two vicinal of R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³ canoptionally be joined to form a carbo- or heterocycle. In someembodiments, Ar¹ is selected from:

In one embodiment, the p38α MAPK inhibitor is a compound of Formula II,and can be selected from any one compound of Formulas 1001-1180 in Table1:

TABLE 1 Compound #

L² Ar¹ 1001

—NHCH₂—

1002

—NHCH₂—

1003

—NHCH₂—

1004

—NHCH₂—

1005

—NHCH₂—

1006

—NHCH₂—

1007

—NHCH₂—

1008

—NHCH₂—

1009

—NHCH₂—

1010

—NHCH₂—

1011

—NHCH₂—

1012

—NHCH₂—

1013

—NHCH₂—

1014

—NHCH₂—

1015

—NHCH₂—

1016

—NHCH₂—

1017

—NHCH₂—

1018

—NHCH₂—

1019

—NHCH₂—

1020

—NHCH₂—

1021

—NHCH₂—

1022

—NHCH₂—

1023

—NHCH₂—

1024

—NHCH₂—

1025

—NHCH₂—

1026

—NHCH₂—

1027

—NHCH₂—

1028

—NHCH₂—

1029

—NHCH₂—

1030

—NHCH₂—

1031 (UM101)

—NHCO—

1032

—NHCO—

1033

—NHCO—

1034

—NHCO—

1035

—NHCO—

1036

—NHCO—

1037

—NHCO—

1038

—NHCO—

1039

—NHCO—

1040

—NHCO—

1041

—NHCO—

1042

—NHCO—

1043

—NHCO—

1044

—NHCO—

1045

—NHCO—

1046

—NHCO—

1047

—NHCO—

1048

—NHCO—

1049

—NHCO—

1050

—NHCO—

1051

—NHCO—

1052

—NHCO—

1053

—NHCO—

1054

—NHCO—

1055

—NHCO—

1056

—NHCO—

1057

—NHCO—

1058

—NHCO—

1059

—NHCO—

1060

—NHCO—

1061

—NHSO₂—

1062

—NHSO₂—

1063

—NHSO₂—

1064

—NHSO₂—

1065

—NHSO₂—

1066

—NHSO₂—

1067

—NHSO₂—

1068

—NHSO₂—

1069

—NHSO₂—

1070

—NHSO₂—

1071

—NHSO₂—

1072

—NHSO₂—

1073

—NHSO₂—

1074

—NHSO₂—

1075

—NHSO₂—

1076

—NHSO₂—

1077

—NHSO₂—

1078

—NHSO₂—

1079

—NHSO₂—

1080

—NHSO₂—

1081

—NHSO₂—

1082

—NHSO₂—

1083

—NHSO₂—

1084

—NHSO₂—

1085

—NHSO₂—

1086

—NHSO₂—

1087

—NHSO₂—

1088

—NHSO₂—

1089

—NHSO₂—

1090

—NHSO₂—

1091

—CH₂NH—

1092

—CH₂NH—

1093

—CH₂NH—

1094

—CH₂NH—

1095

—CH₂NH—

1096

—CH₂NH—

1097

—CH₂NH—

1098

—CH₂NH—

1099

—CH₂NH—

1100

—CH₂NH—

1101

—CH₂NH—

1102

—CH₂NH—

1103

—CH₂NH—

1104

—CH₂NH—

1105

—CH₂NH—

1106

—CH₂NH—

1107

—CH₂NH—

1108

—CH₂NH—

1109

—CH₂NH—

1110

—CH₂NH—

1111

—CH₂NH—

1112

—CH₂NH—

1113

—CH₂NH—

1114

—CH₂NH—

1115

—CH₂NH—

1116

—CH₂NH—

1117

—CH₂NH—

1118

—CH₂NH—

1119

—CH₂NH—

1120

—CH₂NH—

1121

—CONH—

1122

—CONH—

1123

—CONH—

1124

—CONH—

1125

—CONH—

1126

—CONH—

1127

—CONH—

1128

—CONH—

1129

—CONH—

1130

—CONH—

1131

—CONH—

1132

—CONH—

1133

—CONH—

1134

—CONH—

1135

—CONH—

1136

—CONH—

1137

—CONH—

1138

—CONH—

1139

—CONH—

1140

—CONH—

1141

—CONH—

1142

—CONH—

1143

—CONH—

1144

—CONH—

1145

—CONH—

1146

—CONH—

1147

—CONH—

1148

—CONH—

1149

—CONH—

1150

—CONH—

1151

—SO₂NH—

1152

—SO₂NH—

1153

—SO₂NH—

1154

—SO₂NH—

1155

—SO₂NH—

1156

—SO₂NH—

1157

—SO₂NH—

1158

—SO₂NH—

1159

—SO₂NH—

1160

—SO₂NH—

1161

—SO₂NH—

1162

—SO₂NH—

1163

—SO₂NH—

1164

—SO₂NH—

1165

—SO₂NH—

1166

—SO₂NH—

1167

—SO₂NH—

1168

—SO₂NH—

1169

—SO₂NH—

1170

—SO₂NH—

1171

—SO₂NH—

1172

—SO₂NH—

1173

—SO₂NH—

1174

—SO₂NH—

1175

—SO₂NH—

1176

—SO₂NH—

1177

—SO₂NH—

1178

—SO₂NH—

1179

—SO₂NH—

1180

—SO₂NH—

In one embodiment, the p38α MAPK inhibitor is a compound of any ofFormulas 1001 (SF-6-221), 1032 (SF-7-008), SF-7-009, 1034 (SF-7-010),1035 (SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), and 1087 (SF-7-044):

Selective binding of UM101 to p38α was confirmed using complementarytechnologies. DSF, which detects ligand-induced protein stabilizationshowed UM101 to cause a concentration-dependent increase in meltingtemperature of p38α but not p38β (FIG. 3D). The smaller effect of UM101compared with SB203580 on p38α melting suggests lower p38α bindingaffinity of substrate-selective vs. catalytic inhibitors, which issimilar substrate-selective ERK inhibitors. The smaller effect ofSB203580 on p38β than p38α is consistent with the known ˜10-fold higherbinding affinity of SB203580 for p38α. STD-NMR, which measures lowaffinity protein:ligand binding via non-scalar magnetization transferfrom protein to ligand protons, confirmed specific UM101 binding to p38αand localized the interaction to its aromatic rings. UM101 binding toits CADD target was also confirmed by showing that mutating four of tenamino acids in the targeted pocket abrogated UM101 binding whileSB203580 binding was preserved.

In some embodiments, the p38α MAPK inhibitor causes aconcentration-dependent increase in melting temperature of p38α MAPK.The difference in melting temperature ΔTm (° C.) is measured at a p38αMAPK inhibitor concentration of between 1 nM and 1000 μM. In oneembodiment, the difference in melting temperature ΔTm (° C.) is measuredat a p38α MAPK inhibitor concentration of 100 μM. In one embodiment, ΔTmis between about 0.1 and about 2° C. In one embodiment, ΔTm is betweenabout 0.01 and about 0.05° C. In one embodiment, ΔTm is between about0.01 and about 0.1° C. In one embodiment, ΔTm is between about 0.03 andabout 0.7° C. In one embodiment, ΔTm is between about 0.06 and about1.5° C. In one embodiment, ΔTm is between about 1° C. and about 2° C. Inone embodiment, ΔTm is between about 1.5 and about 2° C. In oneembodiment, ΔTm is about 0.1° C. In one embodiment, ΔTm is about 0.2° C.In one embodiment, ΔTm is about 0.3° C. In one embodiment, ΔTm is about0.4° C. In one embodiment, ΔTm is about 0.5° C. In one embodiment, ΔTmis about 0.6° C. In one embodiment, ΔTm is about 0.7° C. In oneembodiment, ΔTm is about 0.8° C. In one embodiment, ΔTm is about 0.9° C.In one embodiment, ΔTm is about 1° C. In one embodiment, ΔTm is about1.1° C. In one embodiment, ΔTm is about 1.2° C. In one embodiment, ΔTmis about 1.3° C. In one embodiment, ΔTm is about 1.4° C. In oneembodiment, ΔTm is about 1.5° C. In one embodiment, ΔTm is about 1.6° C.In one embodiment, ΔTm is about 1.7° C. In one embodiment, ΔTm is about1.8° C. In one embodiment, ΔTm is about 1.9° C. In one embodiment, ΔTmis about 2° C. In one embodiment, ΔTm is about 0.735° C. In oneembodiment, ΔTm is about 0.667° C.

In some embodiments, the p38α MAPK inhibitor has a log P between about−5 and about 10. In some embodiments, the p38α MAPK inhibitor has a logP between about −3 and about 8. In some embodiments, the p38α MAPKinhibitor has a log P between about 0 and about 5. In some embodiments,the p38α MAPK inhibitor has a log P between about 0.1 and about 3. log Pis a measure of drug solubility, and is defined as the logarithm of theoctanol/water partition coefficient of the drug. In one embodiment, thep38α MAPK inhibitor has a log P between about 0.1 and about 1. In oneembodiment, the p38α MAPK inhibitor has a log P between about 0.5 andabout 1.5. In one embodiment, the p38α MAPK inhibitor has a log Pbetween about 0.75 and about 2. In one embodiment, the p38α MAPKinhibitor has a log P between about 1 and about 2.5. In one embodiment,the p38α MAPK inhibitor has a log P between about 1.75 and about 3. Inone embodiment, the p38α MAPK inhibitor has a log P of about 0.1. In oneembodiment, the p38α MAPK inhibitor has a log P of about 0.25. In oneembodiment, the p38α MAPK inhibitor has a log P of about 0.5. In oneembodiment, the p38α MAPK inhibitor has a log P of about 0.75. In oneembodiment, the p38α MAPK inhibitor has a log P of about 1. In oneembodiment, the p38α MAPK inhibitor has a log P of about 1.25. In oneembodiment, the p38α MAPK inhibitor has a log P of about 1.5. In oneembodiment, the p38α MAPK inhibitor has a log P of about 1.75. In oneembodiment, the p38α MAPK inhibitor has a log P of about 2. In oneembodiment, the p38α MAPK inhibitor has a log P of about 2.25. In oneembodiment, the p38α MAPK inhibitor has a log P of about 2.5. In oneembodiment, the p38α MAPK inhibitor has a log P of about 2.75. In oneembodiment, the p38α MAPK inhibitor has a log P of about 3. In oneembodiment, the p38α MAPK inhibitor has a log P of about 0.28. In oneembodiment, the p38α MAPK inhibitor has a log P of about 2.31.

Phosphorylation of MK2 requires binding to the ED site adjacent to theCADD target pocket in p38α MAPK. In some embodiments, the target pocketis at least defined by amino acids R49, H107, L108, and K165 in p38αMAPK. In some embodiments, the target pocket is defined by amino acidsselected from the group consisting of R49, H107, L108, M109, G110, A157,V158, E163, L164, and K165 in p38α MAPK, and combinations thereof. Insome embodiments, the target pocket is defined by the amino acids R49,H107, L108, M109, G110, A157, V158, E163, L164, and K165 in p38α MAPK.Western blotting confirmed partial inhibition of MK2 phosphorylation inanisomycin-stimulated HeLa cells by UM101, but less compared with 10 μMSB203580. SB203580 at a concentration 200- and 20-fold higher than theIC₅₀ for p38α and p38β, respectively failed to completely block MK2phosphorylation, which may reflect a contribution from p38γ or δ as bothisoforms are expressed in HeLa cells.

In one embodiment, the invention relates to a method of inhibiting p38αMAPK where inhibiting p38α MAPK stabilizes an endothelial or epithelialbarrier function. Both of the selective p38α binding compounds, UM60 andUM101, exerted SB203580-like endothelial-barrier-stabilizing andmacrophage-cytokine-modifying effects, thereby validating theED-targeting strategy. UM101 more effectively stabilized endothelialbarriers than SB203580 (FIG. 2A and FIG. 2B) despite having less effecton MK2 phosphorylation. In one embodiment, endothelial barrierpermeability can be measured by separate or combined exposure to TNFαand hyperthermia, followed by measurement of permeability for 10 kDadextran. In one embodiment, endothelial barrier stabilization isassessed by pretreating with a compound of the invention, preceded andfollowed by permeability measurements, where stabilization is expressedas a % reduction in the before and after pretreatment permeabilityincrease. Pretreatment with a p38α MAPK inhibitor can be done at variousconcentrations, for example at 10, 25, 50, or 100 μM. In one embodiment,the permeability increase for 10 kDa dextran can be reduced by between5% to more than 100%. In one embodiment, the permeability increase isreduced by about 5%. In one embodiment, the permeability increase isreduced by about 10%. In one embodiment, the permeability increase isreduced by about 20%. In one embodiment, the permeability increase isreduced by about 30%. In one embodiment, the permeability increase isreduced by about 40%. In one embodiment, the permeability increase isreduced by about 50%. In one embodiment, the permeability increase isreduced by about 60%. In one embodiment, the permeability increase isreduced by about 70%. In one embodiment, the permeability increase isreduced by about 80%. In one embodiment, the permeability increase isreduced by about 90%. In one embodiment, the permeability increase isreduced by about 100%. In one embodiment, the permeability increase isreduced by about more than 100%. In one embodiment, the permeabilityincrease is reduced by about 71%. In one embodiment, the permeabilityincrease is reduced by about 74%. In one embodiment, the permeabilityincrease is reduced by about 89%. In one embodiment, the permeabilityincrease is reduced by about 100%.

Since UM101 more effectively stabilized endothelial barriers thanSB203580 (FIG. 2A and FIG. 2B) despite having less effect on MK2phosphorylation (FIG. 3C), additional molecular actions were evaluatedby comparing the effects of UM101 and SB203580 on global gene expressionusing RNASeq in TNFα-treated HMVECLs. TNFα increased expression of 511genes by ≥2-fold, of which 61 were reduced and 38 increased bypretreatment with 10 μM SB203580. Despite using a concentration of UM101that was >10-fold higher than required to stabilize HMVECL barrierfunctions (FIG. 2A and FIG. 2B), UM101 modified expression of only 38 ofthe 99 SB203580-modified genes. PathwayNet analysis showed UM101 toblock only 7 of the 15 SB203580-blocked transcription factors. MSK1/2was among those spared by UM101, which is consistent with the targetingstrategy for UM101 for the ED site, and in an advantageous way, giventhe anti-inflammatory actions of MSK1/2.

The partial functional overlap of UM101 and SB203580 revealed by RNASeqis consistent with the design of UM101 as a non-catalyticsubstrate-selective inhibitor, but might also be the result ofoff-target effects of SB203580, which include Receptor-interactingProtein Kinase-2, cyclin G-associated kinase, and casein kinase-16.However, none of the SB203580-inhibited transcription factors identifiedby the PathwayNet analysis are known substrates for these kinases asanalyzed using PhosphoNetworks.

Although the high concentration of UM101 used in this analysis may havecaused some p38-independent actions, the data described herein support aconclusion that UM101 exerts its biological effects predominantly bymodifying p38α: (1) DSF and STD-NMR show p38α-specific binding of UM101;(2) p38α binding of UM101 was abrogated by mutating 4 of 10 targetpocket amino acids; (3) UM60 and 101 both bind p38α and exert effects onendothelial function similar to SB203580; (4) UM101 partially blockedphosphorylation of the p38 substrates MK2 and Stat-1 in TNFα-stimulatedHeLa cells; and (5) UM101 inhibited expression of about half the genesinhibited by SB203580. UM101 may be more effective than SB203580 instabilizing endothelial barrier because of its selective sparing ofpotential counter-regulatory genes, such as GM-CSF, MSK1/2-dependentanti-inflammatory genes, and p38ß-dependent pro-survival genes.

In one embodiment, the invention relates to a method of inhibiting p38αMAPK, including contacting the p38α MAPK with a compound capable ofbinding to a pocket near the ED substrate-docking site of p38α MAPK, ora pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. In one embodiment, the compound selectively inhibitsp38α MAPK. In an embodiment, the p38α MAPK inhibitor binds p38α MAPKnear the substrate binding groove of p38α MAPK, which stretches betweentwo acidic patches, the CD and ED domains. In one embodiment, thebinding pocket is defined at least by residues R49, H107, L108, and K165in p38α MAPK. In one embodiment, the binding pocket is defined byresidues R49, H107, L108, M109, G110, A157, V158, E163, L164, and K165in p38α MAPK. In some embodiments, the p38α MAPK inhibitor causes aconcentration-dependent increase in melting temperature of p388a MAPK.In other embodiments, the p388a MAPK inhibitor causes inhibition of MK2phosphorylation. In one embodiment, the compound is of any of FormulasA, I, II, 1001-1180, in particular of any of Formulas 1001 (SF-6-221),1032 (SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037(SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085(SF-6-222), SF-7-009, and 1087 (SF-7-044), or a pharmaceuticallyacceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

In one embodiment, the invention relates to a method of inhibiting p38αMAPK where inhibition of p38α MAPK does not result in loss ofp38α-dependent counterregulatory responses. In some embodiments, thep38α-dependent counterregulatory response relates to mitogen- andstress-activated protein kinase-1 (MSK1), or MSK2. In targeting a pocketnear the ED substrate-docking site of p38α, the inhibitors describedherein avoid interfering with CD-specific substrates, including MSK1/2,thus limiting inflammation through expression of IL-10 and DUSP2. Amongthe effects of MSK1/2 deletion in mice is increased and prolongedLPS-induced expression of the CRP-regulator, IL-6, suggesting a possiblemechanism of the rebound in serum CRP observed in some clinical trialsof catalytic p38 inhibitors.

In one embodiment, the invention relates to a method of inhibiting p38αMAPK where inhibiting p38α MAPK reduces inflammation. In one embodiment,the effects of a p38α MAPK inhibitor on inflammatory cytokine expressionare compared by pretreating PMA-differentiated THP1 cells with a p38αMAPK inhibitor, then stimulating with LPS, and harvesting RNA a periodof time later for analysis by PCR-based cytokine array. In someembodiments, a p38α MAPK inhibitor inhibits expression of various genes,such as IL-1A, IL-8, TNFSF8, CXCL5, CCL7, CCL17, TNFSF9, IL-1B, CXCL1,TNFSF15, CCL5, CCL4, CCL20, CXCL2, TNF, or BMP6. In some embodiments, ap38α MAPK inhibitor inhibits expression of Smad3, which drivesdifferentiation of Foxp3 T regulatory cells and suppressesinterferon-gamma. The p38α MAPK inhibitor can be used in any appropriateconcentration, for example 10, 25, 50, or 100 μM. In one embodiment,inflammation reduction is measured by comparing the fold change mRNAlevels vs. unstimulated PMA-differentiated THP1 cells at variousconcentrations of p38α MAPK inhibitor.

In some embodiments, a p38α MAPK inhibitor modulates TNFα-induced geneexpression in HMVECLs, as evidenced using RNASeq. In one embodiment,HMVECLs were pretreated for a period of time with a p38α MAPK inhibitorat an appropriate concentration, for example 10 μM or 100 μM, and thenstimulated with TNFα for a period of time. A p38α MAPK inhibitor of theinvention inhibits genes such as PRRG4, TSLP, CCL17, EXOC3L4, MMP9,IDO1, CXCL10, CD200, SLC15A3, VDR, IL1B, GPR88, CD207, TCHH, HAS3,GBP1P1, MUC4, ELOVL7, CXCL11, GBP4, PLA1A, or CXCL5.

In one embodiment, the invention relates to a method of inhibiting p38αMAPK where inhibiting p38α MAPK mitigates lung injury in a subject,including, but not limited to, LPS-induced lung injury. In oneembodiment, the effectiveness of a p38α MAPK inhibitor in mitigatingtransalveolar protein and neutrophil extravasation in a mouse model ofLPS/hyperthermia-induced ALI was compared (FIG. 2C and FIG. 2D). In oneembodiment, subjects receive intraperitoneal injection(s) of a p38α MAPKinhibitor at concentrations such as 100, 250, 300, 400, 500, 750, 1000μg, or the like, in an appropriate carrier, for example DMSO, a periodof time prior to intratracheal instillation of LPS, and/or transfer tohyperthermic chambers. Lung lavage from subjects are measured forprotein and/or neutrophils. Compared with control subjects, lavageprotein concentration and neutrophil content in subjects pretreated witha p38α MAPK inhibitor are reduced. In some embodiments, the reduction isbetween about 5% and about 100%. In one embodiment, the reduction isgreater than about 5%. In one embodiment, the reduction is greater thanabout 10%. In one embodiment, the reduction is greater than about 20%.In one embodiment, the reduction is greater than about 30%. In oneembodiment, the reduction is greater than about 40%. In one embodiment,the reduction is greater than about 50%. In one embodiment, thereduction is greater than about 60%. In one embodiment, the reduction isgreater than about 70%. In one embodiment, the reduction is greater thanabout 80%. In one embodiment, the reduction is greater than about 90%.In one embodiment, the reduction is about 100%. In one embodiment, thereduction is less than about 10%. In one embodiment, the reduction isless than about 20%. In one embodiment, the reduction is less than about30%. In one embodiment, the reduction is less than about 40%. In oneembodiment, the reduction is less than about 50%. In one embodiment, thereduction is less than about 60%. In one embodiment, the reduction isless than about 70%. In one embodiment, the reduction is less than about80%. In one embodiment, the reduction is less than about 90%. In oneembodiment, the reduction is about 100%. In one embodiment, thereduction is about 44.1%. In one embodiment, the reduction is about43.9%. In one embodiment, the reduction is about 92.9%. In oneembodiment, the reduction is about 44.4%. In one embodiment, thereduction is about 49.5%. In one embodiment, the reduction is about55.3%. In one embodiment, the reduction is about 54%.

In one embodiment, the invention relates to a method of inhibiting p38αMAPK where inhibiting p38α MAPK regulates leukocyte trafficking.

In one embodiment, the invention relates to a method of inhibiting p38αMAPK where inhibiting p38α MAPK regulates cytokine expression.

Methods of Treatment

The compounds and compositions described herein can be used in methodsfor treating diseases. In some embodiments, the compounds andcompositions described herein can be used in methods for treatingdiseases associated with the up- and/or downregulation of the p38α MAPKprotein.

In one embodiment, the invention relates to a method of treating adisease alleviated by inhibiting the p38α MAPK protein in a patient inneed thereof, including administering to the patient a therapeuticallyeffective amount of a p38α MAPK inhibitor, wherein the p38α MAPKinhibitor is a compound capable of binding to a pocket near the EDsubstrate-docking site of p38α MAPK, or a pharmaceutically acceptablesalt, solvate, hydrate, cocrystal, or prodrug thereof. In oneembodiment, the binding pocket is defined at least by residues R49,H107, L108, and K165 in p38α MAPK. In one embodiment, the binding pocketis defined by residues R49, H107, L108, M109, G110, A157, V158, E163,L164, and K165 in p38α MAPK. In one embodiment, the p38α MAPK inhibitoris a compound of any of Formulas A, I, II, 1001-1180, in particular ofany of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035(SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009 and 1087(SF-7-044), or a pharmaceutically acceptable salt, solvate, hydrate,cocrystal, or prodrug thereof. In some embodiments, the p38α MAPKinhibitor is a p38α MAPK selective inhibitor.

In one embodiment, the invention relates to a method of treating adisease alleviated by inhibiting the p38α MAPK protein in a patient inneed thereof, including administering to the patient a therapeuticallyeffective amount of a p38α MAPK inhibitor in a dosage unit form. In oneembodiment, the dosage unit comprises a physiologically compatiblecarrier medium.

In one embodiment, the invention relates to a method of treating adisease alleviated by inhibiting the p38α MAPK protein in a patient inneed thereof, including administering to the patient a therapeuticallyeffective amount of a p38α MAPK inhibitor, wherein the disease is canceror an inflammatory disease. In some embodiments, the disease isrheumatoid arthritis, a cardiovascular disease, multiple sclerosis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), asthma, acute respiratory distress syndrome (ARDS), or acutelung injury (ALI). In one embodiment, the disease is ahyperproliferative diseases. In some embodiments, the hyperproliferativedisorder is cancer. In some embodiments, the cancer is pancreaticcancer, breast cancer, prostate cancer, lymphoma, skin cancer, coloncancer, melanoma, malignant melanoma, ovarian cancer, brain cancer,primary brain carcinoma, head-neck cancer, glioma, glioblastoma, livercancer, bladder cancer, non-small cell lung cancer, head or neckcarcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma,small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicularcarcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma,colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroidcarcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenalcarcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortexcarcinoma, malignant pancreatic insulinoma, malignant carcinoidcarcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia,cervical hyperplasia, leukemia, acute lymphocytic leukemia, chroniclymphocytic leukemia, acute myelogenous leukemia, chronic myelogenousleukemia, chronic granulocytic leukemia, acute granulocytic leukemia,hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma,polycythemia vera, essential thrombocytosis, Hodgkin's disease,non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primarymacroglobulinemia, or retinoblastoma, and the like. In otherembodiments, the cancer is acoustic neuroma, adenocarcinoma,angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma,bladder carcinoma, brain cancer, breast cancer, bronchogenic carcinoma,cervical cancer, chordoma, choriocarcinoma, colon cancer, colorectalcancer, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma,endotheliocarcinoma, ependymoma, epithelial carcinoma, esophagealcancer, Ewing's tumor, fibrosarcoma, gastric cancer, glioblastomamultiforme, glioma, head and neck cancer, hemangioblastoma, hepatoma,kidney cancer, leiomyosarcoma, liposarcoma, lung cancer,lymphangioendotheliosarcoma, lymphangiosarcoma, medullary carcinoma,medulloblastoma, melanoma, meningioma, mesothelioma, myxosarcoma, nasalcancer, neuroblastoma, oligodendroglioma, oral cancer, osteogenicsarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma,papillary carcinoma, pinealoma, prostate cancer, rabdomyosarcoma, rectalcancer, renal cell carcinoma, retinoblastoma, sarcoma, sebaceous glandcarcinoma, seminoma, skin cancer, squamous cell carcinoma, stomachcancer, sweat gland carcinoma, synovioma, testicular cancer, small celllung carcinoma, throat cancer, uterine cancer, Wilm's tumor, bloodcancer, acute erythroleukemic leukemia, acute lymphoblastic B-cellleukemia, acute lymphoblastic T-cell leukemia, acute lymphoblasticleukemia, acute megakaryoblastic leukemia, acute monoblastic leukemia,acute myeloblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocytic leukemia, acute promyelocytic leukemia, acuteundifferentiated leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, hairy cell leukemia, multiple myeloma, heavy chaindisease, Hodgkin's disease, multiple myeloma, non-Hodgkin's lymphoma,polycythemia vera, or Waldenstrom's macroglobulinemia.

In some embodiments, the hyperproliferative disorder (e.g., cancer)treated by the compounds and compositions described herein includescells having p38α MAPK protein and/or p38α MAPK related proteinexpression.

In one embodiment, the invention relates to a method of treating adisease alleviated by inhibiting the p38α MAPK protein in a patient inneed thereof, including administering to the patient a therapeuticallyeffective amount of a p388a MAPK inhibitor, wherein the p388a MAPKinhibitor is a compound capable of binding to a pocket near the EDsubstrate-docking site of p38α MAPK, or a pharmaceutically acceptablesalt, solvate, hydrate, cocrystal, or prodrug thereof, and one or moreadditional therapeutic agents, including chemotherapeutic and/orimmunotherapeutic agents.

Efficacy of the compounds and combinations of compounds described hereinin treating the indicated diseases or disorders can be tested usingvarious models known in the art, and described herein, which provideguidance for treatment of human disease. Any and all of the describedmethods of treatment may include medical follow-up to determine thetherapeutic or prophylactic effect brought about in the subjectundergoing treatment with the compound(s) and/or composition(s)described herein.

Pharmaceutical Compositions

In an embodiment, an active pharmaceutical ingredient or combination ofactive pharmaceutical ingredients, such as any of the p38α MAPKinhibitors of the invention, is provided as a pharmaceuticallyacceptable composition.

In one embodiment, the invention relates to a pharmaceutical compositionincluding a therapeutically effective amount of a p38α MAPK inhibitorfor the treatment of a disease alleviated by inhibiting p38α MAPKactivity in a patient in need thereof, or a pharmaceutically acceptablesalt, solvate, hydrate, cocrystal, or prodrug thereof; and aphysiologically compatible carrier medium; wherein the p38α MAPKinhibitor is a compound capable of binding to a pocket near the EDsubstrate-docking site of p38α MAPK. In one embodiment, the bindingpocket is defined at least by residues R49, H107, L108, and K165 in p38αMAPK. In one embodiment, the binding pocket is defined by residues R49,H107, L108, M109, G110, A157, V158, E163, L164, and K165 in p38α MAPK.In one embodiment, the p38α MAPK inhibitor is a compound of any ofFormulas A, I, II, 1001-1180, in particular of any of Formulas 1001(SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036(SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061(SF-6-219), 1085 (SF-6-222), SF-7-009 and 1087 (SF-7-044), or apharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. In some embodiments, the p38α MAPK inhibitor is a p38αMAPK selective inhibitor.

In one embodiment, the invention relates to a pharmaceutical compositionincluding a therapeutically effective amount of a p38α MAPK inhibitorfor the treatment of a disease alleviated by inhibiting p38α MAPKactivity in a patient in need thereof, or a pharmaceutically acceptablesalt, solvate, hydrate, cocrystal, or prodrug thereof; and aphysiologically compatible carrier medium, wherein the disease is canceror an inflammatory disease. In one embodiment, the disease is rheumatoidarthritis, a cardiovascular disease, multiple sclerosis, inflammatorybowel disease, chronic obstructive pulmonary disease (COPD), asthma,acute respiratory distress syndrome (ARDS), or acute lung injury (ALI).In one embodiment, the diseases is a cancer such as acoustic neuroma,adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bileduct carcinoma, bladder carcinoma, brain cancer, breast cancer,bronchogenic carcinoma, cervical cancer, chordoma, choriocarcinoma,colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma,embryonal carcinoma, endotheliocarcinoma, ependymoma, epithelialcarcinoma, esophageal cancer, Ewing's tumor, fibrosarcoma, gastriccancer, glioblastoma multiforme, glioma, head and neck cancer,hemangioblastoma, hepatoma, kidney cancer, leiomyosarcoma, liposarcoma,lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, medullarycarcinoma, medulloblastoma, melanoma, meningioma, mesothelioma,myxosarcoma, nasal cancer, neuroblastoma, oligodendroglioma, oralcancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillaryadenocarcinoma, papillary carcinoma, pinealoma, prostate cancer,rabdomyosarcoma, rectal cancer, renal cell carcinoma, retinoblastoma,sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, squamous cellcarcinoma, stomach cancer, sweat gland carcinoma, synovioma, testicularcancer, small cell lung carcinoma, throat cancer, uterine cancer, Wilm'stumor, blood cancer, acute erythroleukemic leukemia, acute lymphoblasticB-cell leukemia, acute lymphoblastic T-cell leukemia, acutelymphoblastic leukemia, acute megakaryoblastic leukemia, acutemonoblastic leukemia, acute myeloblastic leukemia, acute myelomonocyticleukemia, acute nonlymphocytic leukemia, acute promyelocytic leukemia,acute undifferentiated leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, hairy cell leukemia, multiple myeloma, heavy chaindisease, Hodgkin's disease, multiple myeloma, non-Hodgkin's lymphoma,polycythemia vera, or Waldenstrom's macroglobulinemia.

In some embodiments, the concentration of each of the activepharmaceutical ingredients provided in the pharmaceutical compositionsof the invention, such as any of the p38α MAPK inhibitors of theinvention, for example any of the compounds of any of Formulas A, I, II,1001-1180, in particular of any of Formulas 1001 (SF-6-221), 1032(SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037(SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085(SF-6-222), SF-7-009 and 1087 (SF-7-044), is less than, for example,100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%,0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceuticalcomposition.

In some embodiments, the concentration of each of the activepharmaceutical ingredients provided in the pharmaceutical compositionsof the invention, such as any of the p38α MAPK inhibitors of theinvention, for example any of the compounds of any of Formulas A, I, II,1001-1180, in particular of any of Formulas 1001 (SF-6-221), 1032(SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037(SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085(SF-6-222), SF-7-009 and 1087 (SF-7-044), is greater than 90%, 80%, 70%,60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%,18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%,15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%,13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%,10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25%8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25%5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%,2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%,0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%,0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001%w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the concentration of each of the activepharmaceutical ingredients provided in the pharmaceutical compositionsof the invention, such as any of the p38α MAPK inhibitors of theinvention, for example any of the compounds of any of Formulas A, I, II,1001-1180, in particular of any of Formulas 1001 (SF-6-221), 1032(SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037(SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085(SF-6-222), SF-7-009 and 1087 (SF-7-044), is in the range from about0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% toabout 27%, about 0.05% to about 26%, about 0.06% to about 25%, about0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%,about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% toabout 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9%to about 12% or about 1% to about 10% w/w, w/v, or v/v of thepharmaceutical composition.

In some embodiments, the concentration of each of the activepharmaceutical ingredients provided in the pharmaceutical compositionsof the invention, such as any of the p38α MAPK inhibitors of theinvention, for example any of the compounds of any of Formulas A, I, II,1001-1180, in particular of any of Formulas 1001 (SF-6-221), 1032(SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037(SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085(SF-6-222), SF-7-009 and 1087 (SF-7-044), is in the range from about0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%,about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% toabout 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v,or v/v of the pharmaceutical composition.

In some embodiments, the amount of each of the active pharmaceuticalingredients provided in the pharmaceutical compositions of theinvention, such as any of the foregoing p38α MAPK inhibitors of theinvention, for example any of the compounds of any of Formulas A, I, II,1001-1180, in particular of any of Formulas 1001 (SF-6-221), 1032(SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037(SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085(SF-6-222), SF-7-009 and 1087 (SF-7-044), is equal to or less than 10 g,9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g,4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g,0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g,0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or0.0001 g.

In some embodiments, the amount of each of the active pharmaceuticalingredients provided in the pharmaceutical compositions of theinvention, such as any of the p38α MAPK inhibitors of the invention, forexample any of the compounds of any of Formulas A, I, II, 1001-1180, inparticular of any of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034(SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043(SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009,and 1087 (SF-7-044), is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g,0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g,0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g,0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g,0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g,0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g.

Each of the active pharmaceutical ingredients according to the inventionis effective over a wide dosage range. For example, in the treatment ofadult humans, dosages independently range from 0.01 to 1000 mg, from 0.5to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day areexamples of dosages that may be used. The exact dosage will depend uponthe route of administration, the form in which the compound isadministered, the gender and age of the subject to be treated, the bodyweight of the subject to be treated, and the preference and experienceof the attending physician. The clinically-established dosages of thep38α MAPK inhibitors of the invention may also be used if appropriate.

In an embodiment, the molar ratio of two active pharmaceuticalingredients in the pharmaceutical compositions is in the range from 10:1to 1:10, preferably from 2.5:1 to 1:2.5, and more preferably about 1:1.In an embodiment, the weight ratio of the molar ratio of two activepharmaceutical ingredients in the pharmaceutical compositions isselected from the group consisting of 20:1, 19:1, 18:1, 17:1, 16:1,15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1,2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12,1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, and 1:20. In an embodiment,the weight ratio of the molar ratio of two active pharmaceuticalingredients in the pharmaceutical compositions is selected from thegroup consisting of 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1,12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16,1:17, 1:18, 1:19, and 1:20.

In an embodiment, the pharmaceutical compositions described herein, suchas any of the p38α MAPK inhibitors of the invention, for example any ofthe compounds of any of Formulas A, I, II, 1001-1180, in particular ofany of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035(SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087(SF-7-044), are for use in the treatment of an inflammatory disease. Inan embodiment, the pharmaceutical compositions described herein, such asany of the p38α MAPK inhibitors of the invention, are for use in thetreatment of rheumatoid arthritis, a cardiovascular disease, multiplesclerosis, inflammatory bowel disease, chronic obstructive pulmonarydisease (COPD), asthma, acute respiratory distress syndrome (ARDS), oracute lung injury (ALI).

In an embodiment, the pharmaceutical compositions described herein, suchas any of the p38α MAPK inhibitors of the invention, for example any ofthe compounds of any of Formulas A, I, II, 1001-1180, in particular ofany of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035(SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087(SF-7-044), are for use in the treatment of hyperproliferative disordersassociated with the overexpression or up- and/or downregulation p38αMAPK protein. In a some embodiments, the pharmaceutical compositionsdescribed herein are for use in the treatment of a cancer associatedwith overexpression or up- and/or downregulation of p38α MAPK protein,such as pancreatic cancer, breast cancer, prostate cancer, lymphoma,skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer,brain cancer, primary brain carcinoma, head-neck cancer, glioma,glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer,head or neck carcinoma, breast carcinoma, ovarian carcinoma, lungcarcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma,testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomachcarcinoma, colon carcinoma, prostatic carcinoma, genitourinarycarcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiplemyeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma,adrenal cortex carcinoma, malignant pancreatic insulinoma, malignantcarcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignanthypercalcemia, cervical hyperplasia, leukemia, acute lymphocyticleukemia, chronic lymphocytic leukemia, acute myelogenous leukemia,chronic myelogenous leukemia, chronic granulocytic leukemia, acutegranulocytic leukemia, hairy cell leukemia, neuroblastoma,rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essentialthrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissuesarcoma, osteogenic sarcoma, primary macroglobulinemia, orretinoblastoma.

Described below are non-limiting pharmaceutical compositions and methodsfor preparing the same.

Pharmaceutical Compositions for Oral Administration

In an embodiment, the invention provides a pharmaceutical compositionfor oral administration containing the active pharmaceutical ingredientor combination of active pharmaceutical ingredients, such as the p38αMAPK inhibitors described herein, and a pharmaceutical excipientsuitable for oral administration.

In some embodiments, the invention provides a solid pharmaceuticalcomposition for oral administration containing: (i) an effective amountof an active pharmaceutical ingredient or combination of activepharmaceutical ingredients, and (ii) a pharmaceutical excipient suitablefor oral administration. In selected embodiments, the compositionfurther contains (iii) an effective amount of a third activepharmaceutical ingredient, and optionally (iv) an effective amount of afourth active pharmaceutical ingredient.

In some embodiments, the pharmaceutical composition may be a liquidpharmaceutical composition suitable for oral consumption. Pharmaceuticalcompositions of the invention suitable for oral administration can bepresented as discrete dosage forms, such as capsules, sachets, ortablets, or liquids or aerosol sprays each containing a predeterminedamount of an active ingredient as a powder or in granules, a solution,or a suspension in an aqueous or non-aqueous liquid, an oil-in-wateremulsion, a water-in-oil liquid emulsion, powders for reconstitution,powders for oral consumptions, bottles (including powders or liquids ina bottle), orally dissolving films, lozenges, pastes, tubes, gums, andpacks. Such dosage forms can be prepared by any of the methods ofpharmacy, but all methods include the step of bringing the activeingredient(s) into association with the carrier, which constitutes oneor more necessary ingredients. In general, the compositions are preparedby uniformly and intimately admixing the active ingredient(s) withliquid carriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired presentation. Forexample, a tablet can be prepared by compression or molding, optionallywith one or more accessory ingredients. Compressed tablets can beprepared by compressing in a suitable machine the active ingredient in afree-flowing form such as powder or granules, optionally mixed with anexcipient such as, but not limited to, a binder, a lubricant, an inertdiluent, and/or a surface active or dispersing agent. Molded tablets canbe made by molding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent.

The invention further encompasses anhydrous pharmaceutical compositionsand dosage forms since water can facilitate the degradation of somecompounds. For example, water may be added (e.g., 5%) in thepharmaceutical arts as a means of simulating long-term storage in orderto determine characteristics such as shelf-life or the stability offormulations over time. Anhydrous pharmaceutical compositions and dosageforms of the invention can be prepared using anhydrous or low moisturecontaining ingredients and low moisture or low humidity conditions.Pharmaceutical compositions and dosage forms of the invention whichcontain lactose can be made anhydrous if substantial contact withmoisture and/or humidity during manufacturing, packaging, and/or storageis expected. An anhydrous pharmaceutical composition may be prepared andstored such that its anhydrous nature is maintained. Accordingly,anhydrous compositions may be packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastic or the like, unit dose containers,blister packs, and strip packs.

Each of the active pharmaceutical ingredients can be combined in anintimate admixture with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques. The carrier can takea wide variety of forms depending on the form of preparation desired foradministration. In preparing the compositions for an oral dosage form,any of the usual pharmaceutical media can be employed as carriers, suchas, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like in the case of oral liquidpreparations (such as suspensions, solutions, and elixirs) or aerosols;or carriers such as starches, sugars, micro-crystalline cellulose,diluents, granulating agents, lubricants, binders, and disintegratingagents can be used in the case of oral solid preparations, in someembodiments without employing the use of lactose. For example, suitablecarriers include powders, capsules, and tablets, with the solid oralpreparations. If desired, tablets can be coated by standard aqueous ornonaqueous techniques.

Binders suitable for use in pharmaceutical compositions and dosage formsinclude, but are not limited to, corn starch, potato starch, or otherstarches, gelatin, natural and synthetic gums such as acacia, sodiumalginate, alginic acid, other alginates, powdered tragacanth, guar gum,cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch,hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixturesthereof.

Examples of suitable fillers for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.

Disintegrants may be used in the compositions of the invention toprovide tablets that disintegrate when exposed to an aqueousenvironment. Too much of a disintegrant may produce tablets whichdisintegrate in the bottle. Too little may be insufficient fordisintegration to occur, thus altering the rate and extent of release ofthe active ingredients from the dosage form. Thus, a sufficient amountof disintegrant that is neither too little nor too much to detrimentallyalter the release of the active ingredient(s) may be used to form thedosage forms of the compounds disclosed herein. The amount ofdisintegrant used may vary based upon the type of formulation and modeof administration, and may be readily discernible to those of ordinaryskill in the art. About 0.5 to about 15 weight percent of disintegrant,or about 1 to about 5 weight percent of disintegrant, may be used in thepharmaceutical composition. Disintegrants that can be used to formpharmaceutical compositions and dosage forms of the invention include,but are not limited to, agar-agar, alginic acid, calcium carbonate,microcrystalline cellulose, croscarmellose sodium, crospovidone,polacrilin potassium, sodium starch glycolate, potato or tapioca starch,other starches, pre-gelatinized starch, other starches, clays, otheralgins, other celluloses, gums or mixtures thereof.

Lubricants which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to, calciumstearate, magnesium stearate, sodium stearyl fumarate, mineral oil,light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol,other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenatedvegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesameoil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate,ethylaureate, agar, or mixtures thereof. Additional lubricants include,for example, a syloid silica gel, a coagulated aerosol of syntheticsilica, silicified microcrystalline cellulose, or mixtures thereof. Alubricant can optionally be added in an amount of less than about 0.5%or less than about 1% (by weight) of the pharmaceutical composition.

When aqueous suspensions and/or elixirs are desired for oraladministration, the active pharmaceutical ingredient(s) may be combinedwith various sweetening or flavoring agents, coloring matter or dyesand, if so desired, emulsifying and/or suspending agents, together withsuch diluents as water, ethanol, propylene glycol, glycerin and variouscombinations thereof.

The tablets can be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate canbe employed. Formulations for oral use can also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin or olive oil.

Surfactants which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to,hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.That is, a mixture of hydrophilic surfactants may be employed, a mixtureof lipophilic surfactants may be employed, or a mixture of at least onehydrophilic surfactant and at least one lipophilic surfactant may beemployed.

A suitable hydrophilic surfactant may generally have an HLB value of atleast 10, while suitable lipophilic surfactants may generally have anHLB value of or less than about 10. An empirical parameter used tocharacterize the relative hydrophilicity and hydrophobicity of non-ionicamphiphilic compounds is the hydrophilic-lipophilic balance (“HLB”value). Surfactants with lower HLB values are more lipophilic orhydrophobic, and have greater solubility in oils, while surfactants withhigher HLB values are more hydrophilic, and have greater solubility inaqueous solutions. Hydrophilic surfactants are generally considered tobe those compounds having an HLB value greater than about 10, as well asanionic, cationic, or zwitterionic compounds for which the HLB scale isnot generally applicable. Similarly, lipophilic (i.e., hydrophobic)surfactants are compounds having an HLB value equal to or less thanabout 10. However, HLB value of a surfactant is merely a rough guidegenerally used to enable formulation of industrial, pharmaceutical andcosmetic emulsions.

Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionicsurfactants include, but are not limited to, alkylammonium salts;fusidic acid salts; fatty acid derivatives of amino acids,oligopeptides, and polypeptides; glyceride derivatives of amino acids,oligopeptides, and polypeptides; lecithins and hydrogenated lecithins;lysolecithins and hydrogenated lysolecithins; phospholipids andderivatives thereof; lysophospholipids and derivatives thereof;carnitine fatty acid ester salts; salts of alkylsulfates; fatty acidsalts; sodium docusate; acyl-lactylates; mono- and di-acetylatedtartaric acid esters of mono- and di-glycerides; succinylated mono- anddi-glycerides; citric acid esters of mono- and di-glycerides; andmixtures thereof.

Within the aforementioned group, ionic surfactants include, by way ofexample: lecithins, lysolecithin, phospholipids, lysophospholipids andderivatives thereof; carnitine fatty acid ester salts; salts ofalkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono-and di-acetylated tartaric acid esters of mono- and di-glycerides;succinylated mono- and di-glycerides; citric acid esters of mono- anddi-glycerides; and mixtures thereof.

Ionic surfactants may be the ionized forms of lecithin, lysolecithin,phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidic acid, phosphatidylserine, lysophosphatidylcholine,lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidicacid, lysophosphatidylserine, PEG-phosphatidylethanolamine,PVP-phosphatidylethanolamine, lactylic esters of fatty acids,stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides,mono/diacetylated tartaric acid esters of mono/diglycerides, citric acidesters of mono/diglycerides, cholylsarcosine, caproate, caprylate,caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate,linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate,lauroyl carnitines, palmitoyl carnitines, myristoyl carnitines, andsalts and mixtures thereof.

Hydrophilic non-ionic surfactants may include, but not limited to,alkylglucosides; alkylmaltosides; alkylthioglucosides; laurylmacrogolglycerides; polyoxyalkylene alkyl ethers such as polyethyleneglycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethyleneglycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esterssuch as polyethylene glycol fatty acids monoesters and polyethyleneglycol fatty acids diesters; polyethylene glycol glycerol fatty acidesters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fattyacid esters such as polyethylene glycol sorbitan fatty acid esters;hydrophilic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylenesterols, derivatives, and analogs thereof, polyoxyethylated vitamins andderivatives thereof, polyoxyethylene-polyoxypropylene block copolymers;and mixtures thereof; polyethylene glycol sorbitan fatty acid esters andhydrophilic transesterification products of a polyol with at least onemember of the group consisting of triglycerides, vegetable oils, andhydrogenated vegetable oils. The polyol may be glycerol, ethyleneglycol, polyethylene glycol, sorbitol, propylene glycol,pentaerythritol, or a saccharide.

Other hydrophilic-non-ionic surfactants include, without limitation,PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate,PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32distearate, PEG-40 stearate, PEG-100 stearate, PEG-20 dilaurate, PEG-25glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate,PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryllaurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenatedcastor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6caprate/caprylate glycerides, PEG-8 caprate/caprylate glycerides,polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, PEG-30soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitanlaurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearylether, tocopheryl PEG-100 succinate, PEG-24 cholesterol, polyglyceryl-10oleate, Tween 40, Tween 60, sucrose monostearate, sucrose monolaurate,sucrose monopalmitate, PEG 10-100 nonyl phenol series, PEG 15-100 octylphenol series, and poloxamers.

Suitable lipophilic surfactants include, by way of example only: fattyalcohols; glycerol fatty acid esters; acetylated glycerol fatty acidesters; lower alcohol fatty acids esters; propylene glycol fatty acidesters; sorbitan fatty acid esters; polyethylene glycol sorbitan fattyacid esters; sterols and sterol derivatives; polyoxyethylated sterolsand sterol derivatives; polyethylene glycol alkyl ethers; sugar esters;sugar ethers; lactic acid derivatives of mono- and di-glycerides;hydrophobic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids and sterols; oil-solublevitamins/vitamin derivatives; and mixtures thereof. Within this group,preferred lipophilic surfactants include glycerol fatty acid esters,propylene glycol fatty acid esters, and mixtures thereof, or arehydrophobic transesterification products of a polyol with at least onemember of the group consisting of vegetable oils, hydrogenated vegetableoils, and triglycerides.

In an embodiment, the composition may include a solubilizer to ensuregood solubilization and/or dissolution of the compound of the presentinvention and to minimize precipitation of the compound of the presentinvention. This can be especially important for compositions fornon-oral use—e.g., compositions for injection. A solubilizer may also beadded to increase the solubility of the hydrophilic drug and/or othercomponents, such as surfactants, or to maintain the composition as astable or homogeneous solution or dispersion.

Examples of suitable solubilizers include, but are not limited to, thefollowing: alcohols and polyols, such as ethanol, isopropanol, butanol,benzyl alcohol, ethylene glycol, propylene glycol, butanediols andisomers thereof, glycerol, pentaerythritol, sorbitol, mannitol,transcutol, dimethyl isosorbide, polyethylene glycol, polypropyleneglycol, polyvinylalcohol, hydroxypropyl methylcellulose and othercellulose derivatives, cyclodextrins and cyclodextrin derivatives;ethers of polyethylene glycols having an average molecular weight ofabout 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether(glycofurol) or methoxy PEG; amides and other nitrogen-containingcompounds such as 2-pyrrolidone, 2-piperidone, ε-caprolactam,N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone,N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone; esterssuch as ethyl propionate, tributylcitrate, acetyl triethylcitrate,acetyl tributyl citrate, triethylcitrate, ethyl oleate, ethyl caprylate,ethyl butyrate, triacetin, propylene glycol monoacetate, propyleneglycol diacetate, F-caprolactone and isomers thereof, 6-valerolactoneand isomers thereof, β-butyrolactone and isomers thereof; and othersolubilizers known in the art, such as dimethyl acetamide, dimethylisosorbide, N-methyl pyrrolidones, monooctanoin, diethylene glycolmonoethyl ether, and water.

Mixtures of solubilizers may also be used. Examples include, but notlimited to, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate,dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone,polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropylcyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol,transcutol, propylene glycol, and dimethyl isosorbide. Particularlypreferred solubilizers include sorbitol, glycerol, triacetin, ethylalcohol, PEG-400, glycofurol and propylene glycol.

The amount of solubilizer that can be included is not particularlylimited. The amount of a given solubilizer may be limited to abioacceptable amount, which may be readily determined by one of skill inthe art. In some circumstances, it may be advantageous to includeamounts of solubilizers far in excess of bioacceptable amounts, forexample to maximize the concentration of the drug, with excesssolubilizer removed prior to providing the composition to a patientusing conventional techniques, such as distillation or evaporation.Thus, if present, the solubilizer can be in a weight ratio of 10%, 25%,50%, 100%, or up to about 200% by weight, based on the combined weightof the drug, and other excipients. If desired, very small amounts ofsolubilizer may also be used, such as 5%, 2%, 1% or even less.Typically, the solubilizer may be present in an amount of about 1% toabout 100%, more typically about 5% to about 25% by weight.

The composition can further include one or more pharmaceuticallyacceptable additives and excipients. Such additives and excipientsinclude, without limitation, detackifiers, anti-foaming agents,buffering agents, polymers, antioxidants, preservatives, chelatingagents, viscomodulators, tonicifiers, flavorants, colorants, odorants,opacifiers, suspending agents, binders, fillers, plasticizers,lubricants, and mixtures thereof.

In addition, an acid or a base may be incorporated into the compositionto facilitate processing, to enhance stability, or for other reasons.Examples of pharmaceutically acceptable bases include amino acids, aminoacid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide,sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate,magnesium hydroxide, magnesium aluminum silicate, synthetic aluminumsilicate, synthetic hydrocalcite, magnesium aluminum hydroxide,diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine,triethylamine, triisopropanolamine, trimethylamine,tris(hydroxymethyl)aminomethane (TRIS) and the like. Also suitable arebases that are salts of a pharmaceutically acceptable acid, such asacetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonicacid, amino acids, ascorbic acid, benzoic acid, boric acid, butyricacid, carbonic acid, citric acid, fatty acids, formic acid, fumaricacid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lacticacid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionicacid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinicacid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonicacid, uric acid, and the like. Salts of polyprotic acids, such as sodiumphosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphatecan also be used. When the base is a salt, the cation can be anyconvenient and pharmaceutically acceptable cation, such as ammonium,alkali metals and alkaline earth metals. Example may include, but notlimited to, sodium, potassium, lithium, magnesium, calcium and ammonium.

Suitable acids are pharmaceutically acceptable organic or inorganicacids. Examples of suitable inorganic acids include hydrochloric acid,hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, boricacid, phosphoric acid, and the like. Examples of suitable organic acidsinclude acetic acid, acrylic acid, adipic acid, alginic acid,alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boricacid, butyric acid, carbonic acid, citric acid, fatty acids, formicacid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbicacid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid,para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid,salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid,thioglycolic acid, toluenesulfonic acid and uric acid.

Pharmaceutical Compositions for Injection

In some embodiments, a pharmaceutical composition is provided forinjection containing an active pharmaceutical ingredient or combinationof active pharmaceutical ingredients, such as a p38α MAPK inhibitor, forexample any of the compounds of any of Formulas A, I, II, 1001-1180, inparticular of any of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034(SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043(SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009,and 1087 (SF-7-044), and a pharmaceutical excipient suitable forinjection.

The forms in which the compositions of the present invention may beincorporated for administration by injection include aqueous or oilsuspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, orpeanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueoussolution, and similar pharmaceutical vehicles.

Aqueous solutions in saline are also conventionally used for injection.Ethanol, glycerol, propylene glycol and liquid polyethylene glycol (andsuitable mixtures thereof), cyclodextrin derivatives, and vegetable oilsmay also be employed. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, for the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.

Sterile injectable solutions are prepared by incorporating an activepharmaceutical ingredient or combination of active pharmaceuticalingredients in the required amounts in the appropriate solvent withvarious other ingredients as enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, certaindesirable methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Pharmaceutical Compositions for Topical Delivery

In some embodiments, a pharmaceutical composition is provided fortransdermal delivery containing an active pharmaceutical ingredient orcombination of active pharmaceutical ingredients, such as p38α MAPKinhibitors described herein, for example any of the compounds of any ofFormulas A, I, II, 1001-1180, in particular of any of Formulas 1001(SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036(SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061(SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087 (SF-7-044), and apharmaceutical excipient suitable for transdermal delivery.

Compositions of the present invention can be formulated intopreparations in solid, semi-solid, or liquid forms suitable for local ortopical administration, such as gels, water soluble jellies, creams,lotions, suspensions, foams, powders, slurries, ointments, solutions,oils, pastes, suppositories, sprays, emulsions, saline solutions,dimethylsulfoxide (DMSO)-based solutions. In general, carriers withhigher densities are capable of providing an area with a prolongedexposure to the active ingredients. In contrast, a solution formulationmay provide more immediate exposure of the active ingredient to thechosen area.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients, which are compounds that allow increasedpenetration of, or assist in the delivery of, therapeutic moleculesacross the stratum corneum permeability barrier of the skin. There aremany of these penetration-enhancing molecules known to those trained inthe art of topical formulation. Examples of such carriers and excipientsinclude, but are not limited to, humectants (e.g., urea), glycols (e.g.,propylene glycol), alcohols (e.g., ethanol), fatty acids (e.g., oleicacid), surfactants (e.g., isopropyl myristate and sodium laurylsulfate), pyrrolidones, glycerol monolaurate, sulfoxides, terpenes(e.g., menthol), amines, amides, alkanes, alkanols, water, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as polyethylene glycols.

Another exemplary formulation for use in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of an active pharmaceutical ingredient or combination of activepharmaceutical ingredients in controlled amounts, either with or withoutanother active pharmaceutical ingredient.

The construction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art. See, e.g., U.S. Pat.Nos. 5,023,252; 4,992,445; and 5,001,139, the entirety of which areincorporated herein by reference. Such patches may be constructed forcontinuous, pulsatile, or on demand delivery of pharmaceutical agents.

Pharmaceutical Compositions for Inhalation

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra and the p38α MAPK inhibitors described herein, for example any ofthe compounds of any of Formulas A, I, II, 1001-1180, in particular ofany of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035(SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087(SF-7-044). Preferably the compositions are administered by the oral ornasal respiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices that deliver the formulationin an appropriate manner. Dry powder inhalers may also be used toprovide inhaled delivery of the compositions.

Other Pharmaceutical Compositions

Pharmaceutical compositions of the p38α MAPK inhibitors describedherein, for example any of the compounds of any of Formulas A, I, II,1001-1180, in particular of any of Formulas 1001 (SF-6-221), 1032(SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037(SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085(SF-6-222), SF-7-009, and 1087 (SF-7-044), may also be prepared fromcompositions described herein and one or more pharmaceuticallyacceptable excipients suitable for sublingual, buccal, rectal,intraosseous, intraocular, intranasal, epidural, or intraspinaladministration. Preparations for such pharmaceutical compositions arewell-known in the art. See, e.g., Anderson, Philip O.; Knoben, James E.;Troutman, William G, eds., Handbook of Clinical Drug Data, TenthEdition, McGraw-Hill, 2002; and Pratt and Taylor, eds., Principles ofDrug Action, Third Edition, Churchill Livingston, N.Y., 1990, each ofwhich is incorporated by reference herein in its entirety.

Administration of an active pharmaceutical ingredient or combination ofactive pharmaceutical ingredients or a pharmaceutical compositionthereof can be effected by any method that enables delivery of thecompounds to the site of action. These methods include oral routes,intraduodenal routes, parenteral injection (including intravenous,intraarterial, subcutaneous, intramuscular, intravascular,intraperitoneal or infusion), topical (e.g., transdermal application),rectal administration, via local delivery by catheter or stent orthrough inhalation. The active pharmaceutical ingredient or combinationof active pharmaceutical ingredients can also be administeredintraadiposally or intrathecally.

Exemplary parenteral administration forms include solutions orsuspensions of active compound in sterile aqueous solutions, forexample, aqueous propylene glycol or dextrose solutions. Such dosageforms can be suitably buffered, if desired.

Kits

The invention also provides kits. The kits include an activepharmaceutical ingredient or combination of active pharmaceuticalingredients, either alone or in combination in suitable packaging, andwritten material that can include instructions for use, discussion ofclinical studies and listing of side effects. Such kits may also includeinformation, such as scientific literature references, package insertmaterials, clinical trial results, and/or summaries of these and thelike, which indicate or establish the activities and/or advantages ofthe composition, and/or which describe dosing, administration, sideeffects, drug interactions, or other information useful to the healthcare provider. Such information may be based on the results of variousstudies, for example, studies using experimental animals involving invivo models and studies based on human clinical trials. The kit mayfurther contain another active pharmaceutical ingredient. In selectedembodiments, an active pharmaceutical ingredient or combination ofactive pharmaceutical ingredients are provided as separate compositionsin separate containers within the kit. In selected embodiments, anactive pharmaceutical ingredient or combination of active pharmaceuticalingredients are provided as a single composition within a container inthe kit. Suitable packaging and additional articles for use (e.g.,measuring cup for liquid preparations, foil wrapping to minimizeexposure to air, and the like) are known in the art and may be includedin the kit. Kits described herein can be provided, marketed and/orpromoted to health providers, including physicians, nurses, pharmacists,formulary officials, and the like. Kits may also, in selectedembodiments, be marketed directly to the consumer.

In some embodiments, the invention provides a kit comprising acomposition comprising a therapeutically effective amount of an activepharmaceutical ingredient (e.g., a p38α MAPK inhibitor, for example anyof the compounds of any of Formulas A, I, II, 1001-1180, in particularof any of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010),1035 (SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087(SF-7-044)), or combination of active pharmaceutical ingredients or apharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. These compositions are typically pharmaceuticalcompositions. The kit is for co-administration of the activepharmaceutical ingredient or combination of active pharmaceuticalingredients, either simultaneously or separately.

In some embodiments, the invention provides a kit comprising (1) acomposition comprising a therapeutically effective amount of an activepharmaceutical ingredient (e.g., a p38α MAPK inhibitor, for example anyof the compounds of any of Formulas A, I, II, 1001-1180, in particularof any of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010),1035 (SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049(SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087(SF-7-044)), or combination of active pharmaceutical ingredients or apharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and (2) a diagnostic test for determining whether apatient's cancer is a particular subtype of a cancer. Any of theforegoing diagnostic methods may be utilized in the kit.

The kits described above are preferably for use in the treatment of thediseases and conditions described herein. In some embodiments, the kitsare for use in the treatment of an inflammatory disease. In someembodiments, the kits are for use in the treatment of rheumatoidarthritis, a cardiovascular disease, multiple sclerosis, inflammatorybowel disease, chronic obstructive pulmonary disease (COPD), asthma,acute respiratory distress syndrome (ARDS), or acute lung injury (ALI).In a particular embodiment, the kits are for use in the treatment ofhyperproliferative disorders, such as cancer.

In a particular embodiment, the kits described herein are for use in thetreatment of cancer. In some embodiments, the kits described herein arefor use in the treatment of a cancer selected from the group consistingof pancreatic cancer, breast cancer, prostate cancer, lymphoma, skincancer, colon cancer, melanoma, malignant melanoma, ovarian cancer,brain cancer, primary brain carcinoma, head-neck cancer, glioma,glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer,head or neck carcinoma, breast carcinoma, ovarian carcinoma, lungcarcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma,testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomachcarcinoma, colon carcinoma, prostatic carcinoma, genitourinarycarcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiplemyeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma,adrenal cortex carcinoma, malignant pancreatic insulinoma, malignantcarcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignanthypercalcemia, cervical hyperplasia, leukemia, acute lymphocyticleukemia, chronic lymphocytic leukemia, acute myelogenous leukemia,chronic myelogenous leukemia, chronic granulocytic leukemia, acutegranulocytic leukemia, hairy cell leukemia, neuroblastoma,rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essentialthrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissuesarcoma, osteogenic sarcoma, primary macroglobulinemia, andretinoblastoma. In particular embodiments, the kits described herein arefor use in the treatment of malignant melanoma.

Dosages and Dosing Regimens

The amounts of the pharmaceutical compositions administered using themethods herein, such as the dosages of p38α MAPK inhibitors, for exampledosages of any of the compounds of any of Formulas A, I, II, 1001-1180,in particular of any of Formulas 1001 (SF-6-221), 1032 (SF-7-008), 1034(SF-7-010), 1035 (SF-7-011), 1036 (SF-7-012), 1037 (SF-6-217), 1043(SF-6-223), 1049 (SF-6-224), 1061 (SF-6-219), 1085 (SF-6-222), SF-7-009,and 1087 (SF-7-044), will be dependent on the human or mammal beingtreated, the severity of the disorder or condition, the rate ofadministration, the disposition of the active pharmaceutical ingredientsand the discretion of the prescribing physician. However, an effectivedosage is in the range of about 0.001 to about 100 mg per kg body weightper day, such as about 1 to about 35 mg/kg/day, in single or divideddoses. For a 70 kg human, this would amount to about 0.05 to 7 g/day,such as about 0.05 to about 2.5 g/day. In some instances, dosage levelsbelow the lower limit of the aforesaid range may be more than adequate,while in other cases still larger doses may be employed without causingany harmful side effect—e.g., by dividing such larger doses into severalsmall doses for administration throughout the day. The dosage of thepharmaceutical compositions and active pharmaceutical ingredients may beprovided in units of mg/kg of body mass or in mg/m² of body surfacearea.

In some embodiments, the invention includes methods of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress p38α MAPK, the method comprising the steps of administeringa therapeutically effective dose of an active pharmaceutical ingredientthat is a p38α MAPK inhibitor, for example any of the compounds of anyof Formulas A, I, II, 1001-1180, in particular of any of Formulas 1001(SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036(SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061(SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087 (SF-7-044), to the humansubject.

In some embodiments, the invention includes methods of treating a cancerin a human subject suffering from the cancer in which cancer cellsoverexpress p388a MAPK, the method comprising the steps of administeringa therapeutically effective dose of an active pharmaceutical ingredientthat is a p38α MAPK inhibitor, for example any of the compounds of anyof Formulas A, I, II, 1001-1180, in particular of any of Formulas 1001(SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036(SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061(SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087 (SF-7-044), to the humansubject to inhibit or decrease the activity of p38α MAPK protein.

In some embodiments, a pharmaceutical composition or activepharmaceutical ingredient is administered in a single dose. Suchadministration may be by injection, e.g., intravenous injection, inorder to introduce the active pharmaceutical ingredient quickly.However, other routes, including the preferred oral route, may be usedas appropriate. A single dose of a pharmaceutical composition may alsobe used for treatment of an acute condition.

In some embodiments, a pharmaceutical composition or activepharmaceutical ingredient is administered in multiple doses. In anembodiment, a pharmaceutical composition is administered in multipledoses. Dosing may be once, twice, three times, four times, five times,six times, or more than six times per day. Dosing may be once a month,once every two weeks, once a week, or once every other day. In otherembodiments, a pharmaceutical composition is administered about once perday to about 6 times per day. In some embodiments, a pharmaceuticalcomposition is administered once daily, while in other embodiments, apharmaceutical composition is administered twice daily, and in otherembodiments a pharmaceutical composition is administered three timesdaily.

Administration of the active pharmaceutical ingredients may continue aslong as necessary. In selected embodiments, a pharmaceutical compositionis administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 day(s). Insome embodiments, a pharmaceutical composition is administered for lessthan 28, 14, 7, 6, 5, 4, 3, 2, or 1 day(s). In some embodiments, apharmaceutical composition is administered chronically on an ongoingbasis—e.g., for the treatment of chronic effects. In some embodiments,the administration of a pharmaceutical composition continues for lessthan about 7 days. In yet another embodiment the administrationcontinues for more than about 6, 10, 14, 28 days, two months, sixmonths, or one year. In some cases, continuous dosing is achieved andmaintained as long as necessary.

In some embodiments, an effective dosage of an active pharmaceuticalingredient disclosed herein, for example any of the compounds of any ofFormulas A, I, II, 1001-1180, in particular of any of Formulas 1001(SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036(SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061(SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087 (SF-7-044), is in therange of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about20 mg to about 250 mg, about 25 mg to about 200 mg, about 10 mg to about200 mg, about 20 mg to about 150 mg, about 30 mg to about 120 mg, about10 mg to about 90 mg, about 20 mg to about 80 mg, about 30 mg to about70 mg, about 40 mg to about 60 mg, about 45 mg to about 55 mg, about 48mg to about 52 mg, about 50 mg to about 150 mg, about 60 mg to about 140mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90mg to about 110 mg, about 95 mg to about 105 mg, about 150 mg to about250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg,about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mgto about 205 mg, or about 198 to about 202 mg. In some embodiments, aneffective dosage of an active pharmaceutical ingredient disclosed hereinis less than about 25 mg, less than about 50 mg, less than about 75 mg,less than about 100 mg, less than about 125 mg, less than about 150 mg,less than about 175 mg, less than about 200 mg, less than about 225 mg,or less than about 250 mg. In some embodiments, an effective dosage ofan active pharmaceutical ingredient disclosed herein is greater thanabout 25 mg, greater than about 50 mg, greater than about 75 mg, greaterthan about 100 mg, greater than about 125 mg, greater than about 150 mg,greater than about 175 mg, greater than about 200 mg, greater than about225 mg, or greater than about 250 mg.

In some embodiments, an effective dosage of an active pharmaceuticalingredient disclosed herein, for example any of the compounds of any ofFormulas A, I, II, 1001-1180, in particular of any of Formulas 1001(SF-6-221), 1032 (SF-7-008), 1034 (SF-7-010), 1035 (SF-7-011), 1036(SF-7-012), 1037 (SF-6-217), 1043 (SF-6-223), 1049 (SF-6-224), 1061(SF-6-219), 1085 (SF-6-222), SF-7-009, and 1087 (SF-7-044), is in therange of about 0.01 mg/kg to about 200 mg/kg, or about 0.1 to 100 mg/kg,or about 1 to 50 mg/kg.

In some embodiments, an active pharmaceutical ingredient is administeredat a dosage of 10 to 200 mg BID, including 50, 60, 70, 80, 90, 100, 150,or 200 mg BID. In some embodiments, an active pharmaceutical ingredientis administered at a dosage of 10 to 500 mg BID, including 1, 5, 10, 15,25, 50, 75, 100, 150, 200, 300, 400, or 500 mg BID.

In some instances, dosage levels below the lower limit of the aforesaidranges may be more than adequate, while in other cases still largerdoses may be employed without causing any harmful side effect, e.g., bydividing such larger doses into several small doses for administrationthroughout the day. Of course, as those skilled in the art willappreciate, the dosage actually administered will depend upon thecondition being treated, the age, health and weight of the recipient,the type of concurrent treatment, if any, and the frequency oftreatment. Moreover, the effective dosage amount may be determined byone skilled in the art on the basis of routine empirical activitytesting to measure the bioactivity of the compound(s) in a bioassay, andthus establish the appropriate dosage to be administered.

An effective amount of the combination of the active pharmaceuticalingredient may be administered in either single or multiple doses by anyof the accepted modes of administration of agents having similarutilities, including rectal, buccal, intranasal and transdermal routes,by intra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, orally, topically, or asan inhalant.

In some embodiments, the compositions described herein further includecontrolled-release, sustained release, or extended-release therapeuticdosage forms for administration of the compounds described herein, whichinvolves incorporation of the compounds into a suitable delivery systemin the formation of certain compositions. This dosage form controlsrelease of the compound(s) in such a manner that an effectiveconcentration of the compound(s) in the bloodstream may be maintainedover an extended period of time, with the concentration in the bloodremaining relatively constant, to improve therapeutic results and/orminimize side effects. Additionally, a controlled-release system wouldprovide minimum peak to trough fluctuations in blood plasma levels ofthe compound.

The following examples describe the invention in further detail. Theseexamples are provided for illustrative purposes only, and should in noway be considered as limiting the invention.

EXAMPLES Materials and Methods

Chemicals, recombinant proteins and antibodies: Mouse anti-human p38αand rabbit anti-phospho-MK2 (T222) and phospho-Stat-1 (S727) werepurchased from Cell Signaling Technology (Danvers, MA). The codingsequences for human p38α variant 2 and p38p (with N-terminal HA tag)were amplified by PCR and cloned into pRSetA (Thermo Fisher). Mutationswere introduced into p38α using Quikchange (Stratagene) and confirmed bybidirectional sequencing. Plasmids were transformed in E. coli BL21 andproteins were purified using cobalt columns (TALON™; ClontechLaboratories; Mountain View, CA) and confirmed by SDS-PAGE and Westernblotting. The compounds identified in the CADD screen were purchasedfrom Maybridge Chemical Co. (Belgium).

CADD identification of lead compounds (FIG. 1D): Based on the X-raycrystal structure of mouse p38α/MAPK14 (PDB ID: 1P38), a step-wiseiterative CADD process was used to screen an in silico database of smallmolecule compounds available from Maybridge Chemical ScreeningCollection for the potential to bind in a pocket near the ED substratebinding site (FIG. 1A and FIG. 1B). In silico preparation of p38αconformation was performed using CHARMM36 and general (CGenFF) forcefield with the Nanoscale Molecular Dynamics (NAMD) program, to identifylocal potential ligand-binding pockets. Protein structures weresubjected to clustering to identify 20 representative proteinconformations to account for protein flexibility. Screening wasperformed in the following stages: (1) potential inhibitor binding siteswere identified; (2) compounds were ranked based on their van der Waals(VDW) and electrostatic interaction energies with the protein bindingpockets using the program DOCK with size-based score normalization; (3)the top 50,000 compounds were subjected to a second in silico screenwith additional relaxation of the ligands during simulated binding andthe top 1,000 compounds were selected based on total interaction energyincluding score normalization based on ligand size; (4) chemicalfingerprint-based cluster analysis of the top scoring compounds usingthe program MOE (Chemical Computing Group) was performed to identifychemically diverse compounds and the final list of potentialp38α-interacting compounds were selected based on a scalarbioavailability metric, 4DBA, that accounts for the physiochemicaldescriptors in Lipinski's Rule of Five.

Mouse unphosphorylated p38α/MAPK14 variant-1 differs from its humanvariant-2 by only two amino acids, H48L and A263T and from mousevariant-2 and human variant-1 by only 14 amino acids between residues230 and 254. Neither these amino differences nor the phosphorylationstate of p38α (FIG. 1C) are predicted to significantly alter thestructure of the CD or ED sites or our CADD targeted, thereby validatingthe use of mouse unphosphorylated p38α variant-1 for the CADD search andunphosphorylated recombinant human p38α variant-2 protein for the DSFscreen.

Site Identification by Ligand Competitive Saturation (SILCS): An insilico map of all potential ligand-binding pockets in p38α has beencompleted, including the ED site target, using the Site Identificationby Ligand Competitive Saturation (SILCS) method (FIG. 6 , potentialbinding sites in green). The SILCS method creates a free energy map(grid free energy; GFE FragMaps) of the functional group interactionpattern of p38α that allows for identification of putative binding sitesand rapid free energy estimates of ligand binding to the various p38αsites (ligand GFE or LGFE). The SILCS GFE FragMaps account for proteinflexibility, protein desolvation, functional group desolvation, as wellas functional group-protein interactions, thereby yielding highlyaccurate mapping of the protein for use in database screen and leadcompound optimization. Each stepwise in silico CADD screen of anycompound database starts with the SILCS pharmacophore approach, whichtakes into account protein flexibility. The secondary screen is based onthe MC SILCS approach from which relative free energies of binding arecalculated. A final screen based on chemical diversity, physiochemicalproperties that maximize absorption, distribution, metabolism, andexcretion (ADME) characteristics and potential for chemicaloptimization, generates a list of compounds for testing of selectivep38α binding and biological activity. Additional rounds of screening areperformed using CADD strategies modified based on the proteomic andstructural analyses of lead compounds. Searches of the database forstructural analogs of lead compounds from earlier rounds are performedusing the program MOE (Chemical Computing Group).

Alternative CADD methodology: The program Dock can be used with scoringbased on the Dock van der Waals (vdW) interaction energy normalized formolecular weight (MW). This method identifies compounds that stericallyfit the binding site while biasing towards low MW compounds. Additionalranking of compounds use generalized linear response methods and includefree energy of solvation based on the implicit solvent Generalized Born(GB) model.

Alternative p38α targets: the search strategy can be changed to targetthe CD, or the DEF sites. Since formation of the DEF pocket requiresp38α activation, dual-phosphorylated p38α is used for its DSF screen.

Differential Scanning Fluorimetry (DSF): Binding of CADD selectedcompounds to p38α and R isoforms was tested experimentally using DSF,which evaluates changes in the target protein melting temperature (ΔTm)due to interactions with test compound. SYPRO orange (Invitrogen;diluted 1:1000 in 10 mM HEPES, 150 mM NaCl, pH 7.5) and 1 μMunphosphorylated recombinant human p38α were added to 96-well PCRplates, then 50 nM to 200 μM test compound in 100% DMSO (2% final DMSOconcentration) was added, the plates mixed, sealed, centrifuged at 1000rpm for 1 min, and melting curve performed using an Applied Biosystemsreal time PCR instrument. The melting point was determined from thefirst derivative curve. In addition, p38p, or target-disrupted p38αmutant are used as well.

Although DSF is less sensitive than other assays of ligand:proteinbinding, it is low-cost and has relatively high throughput. DSF detectedp38α binding by 25% of the CADD-identified compounds screened andselective p38α binding by 10%, demonstrating good efficiency of both theCADD and DSF screening strategies. The 10% hit rate of the CADD searchfor substrate-selective p38α inhibitors was similar to the search forsubstrate-selective ERK inhibitors, and much greater than the usual0.1-0.01% hit rate using experimental screening alone.

Cell culture: HMVECLs were purchased from Promocell (Heidelberg, DE),maintained in Endothelial Cell Growth Medium MV2, used at passage 3 to10, and studied at postconfluence according to the supplier's protocol.The THP1 human monocyte cell line (American Type Culture Collection/ATCCno. TIB202) was maintained in RPMI 1640 supplemented with 2 mML-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, pH 7.3,penicillin, streptomycin, 0.05 mM β-mercaptoethanol and 10% definedfetal bovine serum (FBS; Gibco, Life Technologies, Grand Island, NY).HeLa cells (ATCC no. CCL-2) were cultured in DMEM with 4.5 g/L glucose,1 mM sodium pyruvate, 2 mM L-glutamine, penicillin, streptomycin, and10% FBS. Prior to experimental exposures, THP1 cells were differentiatedby treating with 5 ng/ml Phorbol 12-myristate 13-acetate (PMA,Sigma-Aldrich) for 24 h, washing with PBS, and culturing at 37° C. inPMA-free media for an additional 24 h.

Endothelial permeability assay: Permeability of HMVECL monolayers wasassessed by measuring transendothelial flux of 10 kDa dextran conjugatedto Cascade blue fluorescent dye for 30 min at 37° C. in Matrigel-coated3 μm pore size Transwell plates.

Cells are treated with test compound at 1-100 μM, 10 μM SB203580, orDMSO for 1 h, then with 10 ng/ml rhTNFα at 39.5° C. for 6 h andpermeability is assessed by adding 100 μg/ml Cascade-blue-conjugated 10kDa dextran to the bottom well for 30 min at 37° C. and analyzingfluorescence (400/420 nm) in the upper well.

Neutrophil transendothelial migration (TEM) assay: Neutrophils wereisolated from heparinized venous blood that was collected from healthyvolunteers using a protocol approved by the University of MarylandInstitutional Review Board and TEM of calcein-labeled neutrophilsthrough HMVECLs was measured.

Cytotoxicity of HMVECL exposed to 10-100 μM of each compound is analyzedby MTS assay (Promega), LDH release (Promega), and immunoblotting foractivated caspase-3 (Cell Signaling).

Macrophage cytokine expression: The capacity of test compounds to blockLPS-induced cytokine expression is assessed in PMA-differentiated THP1cells using qRT-PCR and Luminex-based immunoassays (UMB Cytokine CoreLab). THP1 cells differentiated with 5 ng/ml PMA for 24 h, are treatedwith 1-100 μM test compound, 10 μM SB203580, or DMSO for 1 h, then with100 ng/ml ultrapure E. coli 0111:B4 LPS (InvivoGen) for 3 h (qRT-PCR;Real Time Primers) or 24 h (supernatants for immunoassays).

Mouse acute lung injury Model: Male CD-1 mice weighing 25-30 g werepurchased from Charles River and housed in the Baltimore VeteransAdministration Medical Center Animal Care Facility under AALAC-approvedconditions. All protocols were approved by the University of MarylandBaltimore IACUC. Inhibitors were tested in a mouse i.t. LPS/FRH-inducedALI model. Mice were pretreated with SB203580 or putative p38 inhibitorsin ≤2% DMSO via 0.5 ml i.p. injection 1 h prior to i.t. instillation of50 μg LPS and switch to a 37° C. incubator, which increases coretemperature to ˜39.5° C. Mice were euthanized after 24 h, the lungslavaged with a total of 2 ml PBS, the cells counted and the cell freelavage fluid analyzed for protein content using the Bradford method(Biorad).

Animals are anesthetized with inhaled isoflurane during surgery toimplant intraperitoneal thermistors. Mice receive 0.05-0.1 mg/kgbuprenorphine analgesia s.c. Q12 h for 2 postoperative days. Ifsignificant distress occurs during the ALI model buprenorphine analgesiais administered. LPS is administered in 50 μl PBS via instillation inthe posterior oropharynx during anesthesia with isoflurane. p38inhibitors are administered via i.p. injection with a 25 g needle withthe mouse conscious and lightly restrained.

The combination of FRH and intratracheal LPS induces robust pulmonaryneutrophil influx, cytokine expression, and protein leak by 12-24 h and50% mortality beginning at 48 h. UM101 was more potent than SB203580 inreducing neutrophil and protein accumulation in BAL in this model. Thus,to minimize the number of mice required for this screen, lung injury,lung and extrapulmonary inflammation, and drug toxicity are measured ata single 24 h time point, including BAL protein, neutrophil, andproinflammatory cytokine content, serum levels of IL-6, creatinine andAST (Abcam), and Cardiac Troponin I (MyBiosource). Novel compounds aretested at doses of 4, 12, and 40 mg/kg and compared with vehicle (DMSO)-and SB203580 (40 mg/kg)-treated controls. All vehicle- and drug-treatedmice are exposed to i.t. LPS/FRH and compared with naïve mice. 4 miceper group can be used.

Generally, screening is done in prevention models and final candidatesare also evaluated in a treatment model.

Inhibition of substrate phosphorylation: A functional analysis of UM101to block p38-dependent phosphorylation of MK2 and Stat-1 was performedin HeLa cells. The cells were pre-treated with SB203580 or UM101 for 30min and then activated with 10 μM anisomycin for 30 min. Cell extractsprepared in RIPA buffer containing protease and phosphatase inhibitorswere resolved by SDS-PAGE, transferred to PVDF membrane, blocked with 5%nonfat dry milk, probed with primary antibodies against phosphorylatedMK2 and Stat-1, and total p38α as a loading control. Bands were detectedusing secondary antibodies conjugated to infrared fluorophores andinfrared fluorescence imaging (Odyssey; LICOR).

Cytotoxicity assay: Cytotoxicity was monitored in parallel HMVEC-Lmonolayers established in 96-well culture plates using a colorimetricassay that measured reduction of3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) to a formazan dye (CellTiter 96™; Promega; Madison, WI) accordingto the manufacturer's protocol and quantifying product formation bymeasuring absorption at 490 nm.

Gene Expression: RNA integrity was confirmed by Agilent Bioanalyzer 2100and all samples were confirmed to have RNA integrity scores (RIN) of 10prior to further analysis. Poly(A)-enriched samples were reversetranscribed and sequenced using the Illumina HiSeq platform to generateat least 90 million reads per sample. Intergenic sequence accounted forless than 0.7% of all reads indicating minimal genomic DNAcontamination. Raw data were analyzed using the TopHat read alignmenttool and Homo sapiens genomic reference sequence (Ensembl versionGRCh38.78). Differential gene expression was analyzed using the DESeq Rpackage (Bioconductor) and the negative binomial model. Criteria forsignificant differences in gene expression were (1) false discovery rate(FDR)<0.05, (2) expression level>10^(th) percentile, and (3) ≥2-foldchange. The differential gene expression patterns were further analyzedusing the PathwayNet (Troyanskaya Lab, Princeton) and Ingenuity™ PathwayAnalysis (Qiagen) tools. Cytokine gene expression in THP1 cells wasanalyzed by quantitative RT-PCR using primers in a commerciallyavailable PCR array (HCA-II array; Real Time Primers; Elkins Park, PA)and SYBR-green reaction mix (Biorad), and a BioRad iCycler IQ OpticalModule according to the supplier's protocol. Data were quantified usingthe Gene Expression Ct Difference method, and standardized to levels ofthe housekeeping gene, GAPDH, using Ct values automatically determinedby the thermocycler.

Saturation Transfer Difference Nuclear Magnetic Resonance (STD-NMR): A40 mM stock solution of UM101 was made in d6-DMSO. STD-NMR samplescontained 150 mM NaCl, 50 mM phosphate, pH 7, 200 μM UM101, and 5 μM p38protein in D₂O. Spectra were recorded on an Agilent DD2 500-MHzspectrometer equipped with a 5-mm inverse HFCN probe head at 300 K.During each transient, the protein was saturated with a series of 58GAUSSIAN-shaped pulses (50 ms, 1 ms delay between pulses) using thevendor-supplied STD-ES pulse sequence, for a total saturation time of 3seconds. The on-resonance irradiation of protein was done at 0.5 ppm andoff-resonance irradiation at 30 ppm. The vendor-supplied WATERGATE pulsesequence was used to suppress the water signal in the STD spectrum. Theon-resonance and off-resonance pulse sequences were subtractedinternally. A total of 16,384 transients were collected for each STDexperiment with a 1 second delay between acquisitions, 6000 Hz spectralwidth, and 1.3 second acquisition time.

Comparative proteome and phosphopeptide expression profiling by massspectrometry (MS): Protein expression and the percentage ofphosphorylation of specific proteins is quantified in a label-freemanner using mass spectrometry-based techniques. Specifically, theeffects of compounds of the invention are compared with SB203580 onprotein phosphorylation pattern and proteome expression inTNFα-stimulated HMVECLs and LPS-stimulated THP1 cells using LC-MS/MS.Cells are pretreated for 30 min with 10 μM SB203580 or test compound atEC₅₀ and EC₉₀ (based on HMVECL permeability and THP1 IL-8 expressionassays). For phosphopeptide analysis, cells are stimulated for 0.5, 1.5,and 4 h. Tryptic phosphopeptides are enriched using a commerciallyavailable TiO₂ enrichment protocol (Pierce), then analyzed on a nanoUPLCcoupled Thermo Orbitrap Fusion Tribrid Mass Spectrometer using threestrategies: (1) hybrid Electron-Transfer (ETD)/Higher-Energy Collision(HCD) Dissociation (EThcD); (2) data-dependent decision tree (DDDT)logic; (3) HCD product-dependent ETD (HCD-pd-ETD); and/or (4) nanoUPLCcoupled Waters Synapt G2S Mass Spectrometer, using ion mobility linkedparallel MS (UDMS^(e)). For comparative proteome expression analysis,cells are stimulated for 4 and 12 h and lysates analyzed on the nanoUPLCcoupled Waters Synapt G2S and/or nanoUPLC coupled Thermo Orbitrap FusionTribrid using UDMS^(E) and ADAPT-DDA, respectively. Relative peptideabundance is measured by comparing the MS1 peak area of peptide ions,whose identities and phosphorylation events are confirmed by MS2sequencing using the different fragmentation strategies described above(EThcD, DDDT, HCD-pd-ETD and UDMS^(e)). An aligned AMRT (accurate massand retention time) cluster quantification algorithm as described isused for label free quantification.

Immunoblot analysis: Changes in total in vivo proteome and thephosphoproteome are confirmed by immunoblotting using commercialantibodies and infrared fluorescence imaging (Odyssey; LICOR). In vitrokinase assays is performed in reactions containing recombinant activep38α and one or more recombinant substrate proteins and analyzed byimmunoblotting with phosphospecific antibodies.

X-ray crystallography: Higher resolution analysis of compound binding top38α is provided by x-ray crystallography. The primary approach togrowing p38α includes co-crystallization of compound crystals with a 2:1compound:p38α molar ratio. Alternatively, compounds are soaked intopreformed p38α crystals. Diffraction quality protein crystals are grownand screened using an automated system comprising an Alchemist DT screenmaker, Gryphon drop setter with LCP module, and Minstrel DT UV/Visautomated visualization system (Rigaku). These structures are solved bymolecular replacement methods using known p38α structures and standardcrystallographic analysis software (SBGrid).

Analysis of p38-binding kinetics: The KD for the compounds of theinvention are estimated by DSF. ITC is performed to refine the KDcalculation of compounds and produce thermodynamic information tofacilitate ligand optimization. Data is collected on an Auto ITC HTMicrocalorimeter (MicroCal). Recombinant p38α (10 μM) and stockconcentration of test compounds (200 μM) are prepared in identicalbuffers containing low ionization energy (e.g., 50 mM phosphate orcitrate with 50 mM NaCl) and degassed. Heat generation/absorption duringtitration of compound are measured and analyzed with MicroCal software.

Pharmacokinetic/pharmacodynamic (PK/PD) analysis of lead compounds:Compounds are comprehensively analyzed for in vivo toxicity andeffectiveness as both prevention and treatment in the intratrachealLPS+FRH-induced mouse ALI model. This model is a short-term model ofhuman ARDS, amenable to parenteral dosing of therapeutic agents,characterized by extensive endothelial permeability, neutrophilaccumulation, proinflammatory cytokine and chemokine expression,epithelial injury, and ˜50% mortality beginning after 48 h. The resultsare generalizable to other inflammatory diseases. Compounds aresolubilized in a final concentration of ≤1% DMSO and administered as asingle intraperitoneal injection. Maximal tolerated dose (MTD) isdetermined by monitoring mice for 24 h for signs of distress (includingchange in locomotor activity, weight loss, reduced grooming, and ruffledfur), creatinine, BUN, aspartate transaminase (AST), and cardiactroponin. Inhibitors are administered either 30 min prior to or 8 hafter LPS+FRH as models of prevention or treatment, respectively.

Animal number and sex: All testing is performed in CD1 mice, a robuststrain in which ALI and pneumonia models have been validated. Doseescalation uses 2 mice per dose and 24 h observation according topublished guidelines. Survival differences are tested in groups of 20mice (to detect differences in survival of 75% vs. 25%; α=0.05; β=0.2).Group sizes of 6 mice for BAL and plasma analysis of injury/inflammationand lung homogenates for analysis of apoptotic signaling and groups of 4mice for histology are used. Survival experiments are performed in equalnumbers of male and female mice and differences compared by 2-way ANOVA.Additional experiments are added to analyze any unexpected genderdifferences found in drug effects. In some embodiments, experiments willuse male mice.

Maximal Tolerated Dose of most potent, structurally distinct compoundsis determined by measuring toxicity of 20, 40, and 80 mg/kg i.p. in 2mice per dose, monitored for 24 h and euthanized. Serum is analyzed formarkers of hepatic, renal, and cardiac toxicity. Kidney, heart, liverand lung (inflated) are fixed, paraffin-embedded, H&E-stained, andexamined for inflammation and injury. Control mice receive vehicle (1%DMSO). Toxic compounds are replaced by the next structurally distinctcompound on the list of candidates.

Activity of Inhibitors in blocking FRH augmented LPS-induced ALI:Compounds are tested at the MTD in the LPS+FRH-induced ALI model.

Effect of pretreatment on survival: Effectiveness of pretreatment withtest compounds at the MTD on survival in LPS/FRH-challenged mice iscompared with 40 mg/kg SB203580 and vehicle (1% DMSO) in groups of 20mice. Mice receive pretreatment as a single 0.5 ml injection and 30 minlater receive 50 μg LPS via i.t. instillation and placed in 37° C.ambient temperature. This exposure increases core temperature from 36.5°C. to 39.5° C., but is confirmed in some mice using telemetrictemperature monitoring (Data Sciences International; St. Paul, MN). Miceare monitored for survival using moribundity as a surrogate for death.Those compounds showing survival advantage vs. DMSO are further analyzedfor efficacy when given 24 h post-LPS. Ineffective compounds arereplaced by the next compound from the candidate list. Effectivecompounds are further tested at 10% and 30% of the MTD.

Effect of post-LPS dosing on survival: Compounds that are effective aspretreatment are analyzed for effectiveness at the same doses using thesame protocol except delaying dosing until 8 h after LPS instillationand initiation of FRH. Compounds conferring survival advantage vs.SB203580 are analyzed for biological effects and PK. Ineffectivecompounds are replaced by the next compound on the list.

Effect of compounds on inflammation, lung injury, and permeability: Themost effective compounds in the survival experiments are furtheranalyzed for effects on lung injury and inflammation in the LPS+FRH ALImodel. Mice are pretreated with each compound at its ED50 based on thesurvival experiments, 40 mg/kg SB203580, or DMSO 30 min prior to or 8h-after LPS/FRH challenge and euthanized 24 h post-LPS. In 6 mice pergroup, BALF is collected and analyzed for neutrophil content by countingmodified-Giemsa-stained cytopreps, total protein by Bradford method, andlevels of cytokines by Luminex-based immunoassay (UMB Cytokine CoreLab). After lavage, lungs are excised, snap-frozen in liquid nitrogenand homogenates prepared for immunoblotting of candidate p38α substratesto confirm substrate inhibitor effects found in vitro. Lungs from 4 miceper group are inflation/fixed at 20 cm H₂O with Prefer™, paraffinembedded, H&E stained, or GR-1 immunostained to analyze lung injury andneutrophil infiltration, and TUNEL staining and immunostaining foractive caspase-3 to assess apoptosis. Serum IL-6 is measured as anindicator of systemic inflammation.

Pharmacokinetics of novel p38 modifiers: PK of effective compounds inmice is characterized. First, a bioanalytical method for each compoundis developed and validated according to FDA Guidance. PK studies arethen conducted to determine lung uptake and key PK parameters thatcharacterize each compound (i.e., clearance (CL), volume of distribution(Vd), maximum plasma conc. (C_(max)), time to reach C_(max) (T_(max)),area under the plasma concentration curve (AUC) and half-life(t_(1/2))). The PK parameters are used to estimate the time needed toreach steady state plasma concentration (equivalent to five half-lives),and to guide dose selection for further PD studies. In addition, thesestudies help rank the tested p38 modifiers in terms of their lung/plasmaconc. ratios. For each study, CD1 mice (n=30) are treated with a singlei.p. dose (10-50 mg/kg) of the selected p38 modifier (dose range foreach p38 modifier is dependent on the outcome of studies outlined above.In some embodiments, mice (n=3/time point) are euthanized at a pre-doseand at 5, 15, 30, 60, 120, 240, 360, 600, 720 min post dose. Blood andlung samples are analyzed using validated HPLC methods.

Data Analysis: Pathways modified by compounds of the invention ascompared with SB203580 are deduced-by: (1) analyzing comparativeproteome expression using Ingenuity Pathway Analysis and PathwayNet,similarly to RNASeq data from UM101; and (2) analyzing the comparativephosphoproteome by quantitative approaches and bioinformatics. The massspectrometry results are confirmed by analyzing phosphorylation ofcandidate substrates in cells and in vitro kinase assays byimmunoblotting. Off-target binding suggested by the proteomics data isevaluated over a broad concentration range of test compound by DSF andSTD-NMR and by phosphoimmunoblotting for specific substrates andconfirmed in in vitro kinase reactions. By identifying common pathwaysmodified by multiple lead compounds and their interactions with p38α,the common p38α effects required for their favorable biological activityare deduced and incorporated into the CADD algorithm for subsequentsearches and lead compound optimization.

Since the objective of this invention is, in one embodiment, to identifyand characterize the PD/PK properties of novel anti-inflammatorycompounds, these compounds are tested in one embodiment, in order ofactivity based on functional screens, and compounds that fail toxicityor efficacy in survival studies are replaced with the next most potentand structurally dissimilar compound. Compounds are compared withvehicle alone and SB203580 using one-way ANOVA/Fisher PLSD. The PK datais analyzed by the naive averaging method. Compartmental modeling isused to estimate various pharmacokinetic parameters using Phoenixplatform (ver. 1.3, Pharsight, Sunnyvale, CA). Several compartmentalmodels are evaluated to determine the best-fit model. A variety ofweighting schemes are used including equal weight, 1/y, 1/y{circumflexover ( )}, 1/y², and 1/y{circumflex over ( )}², where y is the observeddrug conc., and y{circumflex over ( )} is the model-predicted drug conc.In some embodiments, a final model is selected based on goodness-of-fitplots, weighted residual sum of squares, random distribution ofresiduals, precision of parameter estimates, Akaike's informationcriteria, and Schwarz criteria. After the final model is developed,estimates of the PK parameters are reported including plasma CL, Vd,C_(max), T_(max), AUC, and t_(1/2). Lung uptake is represented as alung/plasma (L/P) conc. ratio.

Alternative Approaches: If phosphospecific antibodies are not availableand phosphorylation does not cause detectable shifts on immunoblots,cell lysates can be enriched using TiO2 prior to immunoblotting.Incubation times can be adjusted, as needed, based on in vitro and invivo proteomics and immunoblot results. Low protein abundance couldpreclude phosphoprotein detection in cell lysates despite maximumstarting material or using isolated cell fractions. In this case, the invivo cellular phosphoproteome analysis can be augmented by usingLC-MS-MS to comprehensively analyze the effects of inhibitors onphosphopeptide patterns in p38α in vitro kinase assays using celllysates as substrate after inactivating endogenous kinases with5′-4-fluorosulphonylbenzoyladenosine (FSBA). Stable isotope dimethyllabeling can be used in case of ambiguous label-free results. Otherback-up technologies include deuterium-hydrogen exchange massspectroscopy and NMR, and DSF/STD-NMR-assessed binding to wild-type p38αand CADD-target-mutants. Surface plasmon resonance (SPR) (Biacore T200Core) can be evaluated as an alternative to ITC to reduce theprotein/compound requirements.

Statistical methods: Data are presented as mean±SE. Differences among >2groups were analyzed by applying a Tukey Honestly Significant Differencetest to a one-way analysis of variance (ANOVA). Differences betweendose-response curves was analyzed by multivariate ANOVA (MANOVA)Differences with p<0.05 were considered significant.

Example 1: CADD Modeling of p38 MAPK Substrate-Docking Site, CompoundIdentification, and Screening Compounds for Direct, SelectiveInteraction with p38α

The inhibitors and methods of the invention relate to a CADD-basedstrategy to identify low molecular weight compounds predicted to bindnear the ED substrate-docking site of mouse unphosphorylated p38α(MAPK14 variant-1; PDB:1P38), which is >99% identical with human p38α(variant-2) (FIG. 1A). The ED and CD sites in p38α are located at eitherend of a substrate-binding cleft located on the opposite side of theprotein from the catalytic site (FIG. 1A). A pocket near the ED bindingsite comprising 10 amino acids, only 7 of which were identical in p38αand p380, was identified (FIG. 1 ). Overlay of structures of mouseunphosphorylated (PDB:1P38) and dual-phosphorylated p38α (PDB:3PY3)revealed near-superimposition of the targeted pocket in the two forms(FIG. 1C).

An overview of the CADD screening and compound testing protocols isshown in FIG. 1D. The compounds in the Maybridge Screening Collectionwere analyzed for binding to the targeted p38α pocket based on Van DerWaals (VDW) and electrostatic interaction energies, chemical diversityby chemical fingerprint-based cluster analysis, solubility, molecularweight and number of hydrogen bonding functional groups that maximizebioavailability.

Twenty structurally dissimilar compounds were selected for functionalanalysis (Table 2), out of a panel of 150 diverse compounds (Table 3)which were selected for potential biological testing.

TABLE 2 CADD-identified p38α ED site-binding candidates screened forp38α binding CADD p38α ΔTm (° C.) ERK2 ΔTm (° C.) no. Compound ID¹ MWlogP² @100 μM³ @100 μM³ 2 SEW 06373 417 3.19 −0.05 0.412 3 HTS 02798 4150.67 0.282 0.337 4 HTS 13333 312 −1.10 0.065 0.452 5 SCR 00846 418 2.220.808 0.628 8 AW 00509 317 1.13 −0.07 0.531 13 SEW 06264 309 0.28 0.0050.390 16 SCR 00610 339 1.69 −0.052 0.444 23 SCR 01200 378 2.79 −0.488−0.598 29 BTB 05645 350 3.07 −0.353 0.342 31 KM 04113 304 1.83 −0.2780.153 43 CD 11992 300 1.16 −0.485 0.151 55 SP 01164 2.11 1.92 −0.5060.022 60 BTB 13869 426 0.28 0.735 0.195 63 PD 00612 294 0.61 −0.2870.075 69 KM 00081 345 1.68 −0.233 0.361 101 HTS 05732 378 2.31 0.6670.0175 115 NRB 03986 278 3.88 −0.156 0.246 141 SEW 02182 318 2.46 0.5540.238 146 KM 10445 313 2.55 −1.084 −1.632 150 HTS 03239 341 1.68 −0.1710.133 ¹Compound ID from Maybridge portfolio. ²logP is the logarithm ofthe estimated octanol/water partition coefficient, a measure of compoundsolubility ³Change in melting temperature relative to DMSO control inDSF assay

TABLE 3 Top 150 CADD-identified p38α ED site-binding candidates CADD no.Compound ID¹ MW logP² 1 AW 1221 442 3.84 2 SEW 06373 417 3.19 3 HTS02798 415 0.67 4 HTS 13333 312 −1.10 5 SCR 00846 418 2.22 6 HTS 01830400 4.15 7 KM11105 409 1.27 8 AW 00509 317 1.13 9 SCR 01457 401 2.12 10KM 09878 362 2.45 11 BTB 10384 434 2.32 12 HTS 03243 419 3.46 13 SEW06264 309 0.28 14 CD 06142 382 3.29 15 KM 08516 382 2.03 16 SCR 00610339 1.69 17 KM 09250 364 0.87 18 SCR 01462 344 −0.25 19 KM 08262 3751.34 20 SCR 01164 430 3.44 21 HTS 05992 360 2.65 22 CD 00735 390 1.72 23SCR 01200 378 2.79 24 SCR 01160 390 0.69 25 SCR 00883 398 2.09 26 AW01002 331 1.49 27 KM 10346 339 1.52 28 KM 09924 374 2.25 29 BTB 05645350 3.07 30 HTS 01722 401 3.5 31 KM 04113 304 1.83 32 SCR 00662 338 2.6233 RJC 02765 348 1.21 34 HTS 08093 330 0.50 35 KM 09335 352 1.08 36 HTS06913 355 1.52 37 KM 07646 296 0.23 38 KM 06447 355 2.44 39 HTS 01903444 2.51 40 KM 06789 333 1.38 41 EN 00285 380 2.34 42 JFD 01748 321 2.7443 CD 11992 300 1.16 44 KM 03098 455 2.56 45 RJF 01988 450 3.99 46 RH00635 402 4.14 47 GK 02919 363 1.17 48 KM 02331 451 3.96 49 GK 01789 3602.91 50 GK 03735 376 1.38 51 HTS 05862 364 1.97 52 KM 07197 337 0.40 53BTB 02067 305 1.94 54 JFD 01679 357 3.55 55 SP 01164 2.11 1.92 56 KM00730 450 1.92 57 HTS 03184 407 3.33 58 HTS 01701 397 4.06 59 HTS 11459409 −1.37 60 BTB 13869 426 0.28 61 RJC 00192 360 3.85 62 HTS 06577 3673.73 63 PD 00612 294 0.61 64 HTS 09813 453 2.98 65 RJC 02517 404 1.93 66DP 01615 356 4.00 67 DP 01320 385 3.74 68 JFD 01765 352 3.24 69 KM 00081345 1.68 70 RDR 03171 419 2.14 71 HTS 04127 398 2.82 72 AW 00409 4032.36 73 BTB 06009 413 2.14 74 KM 10383 443 2.81 75 HTS 05233 369 0.82 76KM 05297 428 0.00 77 CD 11533 373 3.22 78 KM 04839 441 3.01 79 CD 09639460 3.00 80 HTS 04160 414 2.73 81 KM 07794 358 3.70 82 CD 04864 420 3.5183 RDR 02594 397 3.10 84 DP 01806 435 3.43 85 HTS 03190 388 3.29 86 KM09808 405 3.70 87 CD 09308 396 2.27 88 SPB 01817 416 3.99 89 KM 07150411 2.05 90 KM 09339 381 0.91 91 RDR 01132 415 3.32 92 SS 00046 322 3.6393 HTS 02914 351 1.98 94 KM 02270 381 4.08 95 CD 09636 366 1.15 96 KBK00012 364 3.69 97 HTS 13527 337 0.78 98 BB 06821 389 3.99 99 AW 01218343 2.37 100 PD 00703 303 0.33 101 HTS 05732 378 2.31 102 HTS 03187 3570.79 103 HTS 05493 427 1.73 104 RJF 01945 356 3.81 105 CD 05416 378 3.30106 CD 08365 285 1.37 107 SPB 02947 372 3.15 108 SCR 01004 357 0.95 109HTS 05491 429 3.03 110 HTS 02224 372 0.33 111 KM 05869 421 1.45 112 KM02112 388 3.19 113 KM 07452 347 0.49 114 RJC 02844 302 2.65 115 NRB03986 278 3.88 116 SEW 06625 373 3.05 117 SCR 0170 320 −0.70 118 SPB06098 373 4.07 119 FM 00079 382 3.19 120 BTB 03095 350 1.91 121 KM 08272382 1.99 122 BTB 07326 458 3.97 123 HTS 10719 386 3.71 124 JFD 01751 3751.21 125 HTS 05737 366 0.34 126 BTB 02557 300 −0.17 127 KM 01947 3863.26 128 KM 04674 340 2.99 129 BTB 14836 358 1.88 130 KM 07275 346 3.43131 RH 02254 321 1.27 132 S 07734 274 2.06 133 KM 03963 308 2.90 134 KM01163 377 2.95 135 SEW 05535 324 −1.08 136 RDR 02622 321 2.97 137 AW00695 338 −0.37 138 RJC 03556 323 1.30 139 SP 00787 415 2.74 140 JFD02020 322 0.84 141 SEW 02182 318 2.46 142 SEW 00427 350 1.64 143 HTS00966 311 3.43 144 HTS 02841 339 −0.16 145 KM 06585 371 2.32 146 KM10445 313 2.55 147 KM 03965 356 3.97 148 AW 00554 345 0.36 149 HTS 01470371 2.01 150 HTS 03239 341 1.68 ¹Compound ID from Maybridge portfolio.²logP is the logarithm of the octanol/water partition coefficient, ameasure of drug solubility.

Test compounds at 10-100 μM were screened for binding to recombinantp38α and ERK2 using DSF (FIG. 1E, Table 1). Five compounds causedconcentration-dependent stabilization of p38α, indicating binding. Threeof these also stabilized ERK2 (3, 5, and 141 highlighted yellow (with“*”)) and two (highlighted blue (with “+”)), UM60(N2,N7-di(2-hydroxyethyl)-9-oxo-9H-2,7-fluorenedisulfonamide) and UM101(4-chloro-N-{4-[(1,1-dioxo-1lambda-6-,4-thiazinan-4-yl)methyl]phenyl}benzamide),stabilized p38α but not ERK2. These two structurally dissimilarcompounds (FIG. 1F), added at 100 μM, increased the melting temperatureof p38α by ˜0.7° C., compared with a 6° C. increase with SB203580.

MC SILCS docking of UM101 to the ED site and GFE FragMap analysis hasidentified several structural features that can be modified to improveselectivity and potency (FIG. 7 ). The modifiable sites on UM101correspond to those identified as interacting with the p38α in the NMRSTD analysis (FIGS. 3F-3K).

Example 2: Effects of Compounds on Endothelial Barrier Functions

The capacity of UM60 and UM101 to stabilize endothelial barriers tomacromolecules and neutrophils in TNFα- and hyperthermia-stressed HMVECLmonolayers was tested (FIGS. 2A-2D). Combined exposure to 1 ng/ml TNFαand hyperthermia (39.5° C.) for 6 h increased permeability for 10 kDadextran 2.8-fold, compared with untreated 37° C. cells. Pretreating with10 μM SB203580 for 30 min reduced TNFα/hyperthermia-induced permeabilityby 50% (FIG. 2A). Pretreatment with UM60 at 10 and 25 μM had no effecton permeability, but 100 μM UM60 reduced the TNFα/hyperthermia-inducedpermeability increase by 71% while UM101 at 10, 25, and 100 μM reducedthe TNFα/hyperthermia-induced permeability increase by 74%, 89%and >100%, respectively.

Preincubating HMVECLs at 39.5° C. for 6 h increased subsequentIL-8-directed neutrophil TEM from 22.8±0.45×10³ to 31.8±0.54×10³neutrophils (FIG. 2B). Pretreatment with 10 μM SB203580 reducedhyperthermia-augmented neutrophil TEM by 84%. UM60 at 10 and 25 μM andUM101 at 10 μM reduced hyperthermia-augmented increase in TEM by 18%,89%, and 95%. UM60 at 50 μM and UM101 at 25 and 50 μM reduced TEM toless than baseline levels. Neither compound was toxic in LDH release andMTS assays when added to HMVECLs at 100 μM for 48 h.

Example 3: Comparing Effectiveness of SB203580 and UM101 in Mouse ALI

The effectiveness of UM60, UM101, and SB203580 in mitigatingtransalveolar protein and neutrophil extravasation in a mouse model ofLPS/hyperthermia-induced ALI was compared (FIG. 2C and FIG. 2D). Micereceived a single intraperitoneal injection of 100, 300, 500, or 1000 μgUM101, 1000 μg UM60, or 1000 μg SB203580 in 0.5 ml 2% DMSO 30 min priorto intratracheal instillation of 50 μg LPS and transfer to hyperthermicchambers. Control mice received DMSO. Four of six UM60-treated, one ofsix SB203580-treated, and one of eleven DMSO-treated control mice diedwithin 24 h. All sixteen UM101-pretreated mice survived. Lung lavagefrom DMSO-pretreated, LPS/hyperthermia-challenged mice contained1.09±0.19 mg/ml protein and 3.97±1.07×10⁶ neutrophils. Compared withDMSO-treated controls, lavage protein concentration and neutrophilcontent in mice pretreated with 1000 μg SB203580 were reduced by 42% and46.8%, respectively. Lavage protein concentration in mice pretreatedwith 100 μg, 300 μg, 500 μg, and 1000 μg UM101 was reduced by 0, 44.1%,43.9%, and 92.9%, respectively and lavage neutrophil content was reducedby 44.4%, 49.5%, 55.3 and 54%, respectively.

Example 4: Effect of SB203580 and UM101 on LPS-Induced Gene Expressionin Human THP1 Promonocytes

The effects of UM101 and SB203580 on inflammatory cytokine expressionwere compared by pretreating PMA-differentiated THP1 cells with 25 μMSB203580 or 10, 25, or 100 μM UM101 for 30 min, then stimulating with100 ng/ml LPS, and harvesting RNA 4 h later for analysis by PCR-basedcytokine array. Of 16 LPS-stimulated genes in the array, SB203580inhibited expression of seven, IL-1α, IL-8, TNFSF8 (CD30 ligand), TNFSF9(CD137 ligand), CXCL5, CCL7, and CCL17 (Table 4). UM101 inhibitedexpression of all SB203580-inhibited genes except TNFSF9, and inhibitedfour SB203580-insensitive genes, IL-1β, CXCL1, TNFSF15, and CCL5.

TABLE 4 Effects of SB203580 and UM101 on LPS-induced cytokine expressionin THP1 cells¹ SB203580 P vs. UM101 P vs. UM101 P vs. UM101 P vs. GeneDMSO² ANOVA³ 25 μM LPS⁴ 10 μM LPS⁴ 25 μM LPS 100 μM LPS⁴ IL-1A 453 ± 24<0.0001  141 ± 9.2 <0.0001  424 ± 22.6 0.74  339 ± 13.5 0.041  88 ± 3.33<0.0001 IL-8 56.5 ± 3.3 0.0026  9.6 ± 0.1 0.002 35.6 ± 0.7 0.40 26.7 ±4.1  0.564 19.7 ± 1.8  0.015 TNFSF8 60.5 ± 5.5 0.0073 20.6 ± 8.8 0.02423.5 ± 8.3 0.37 10.5 ± 3.9  0.006 20.7 ± 9.5  0.025 CXCL5 49.7 ± 2.9<0.0001  3.2 ± 1.0 <0.0001 23.2 ± 3.7 0.0002 8.7 ± 2.9 <0.0001 3.1 ± 0.2<0.0001 CCL7 12.8 ± 1.2 <0.0001  4.2 ± 0.3 <0.0001  7.7 ± 0.3 0.0036 6.2± 0.9 0.0036  4 ± 0.4 <0.0001 CCL17 56.9 ± 6.1 <0.0001 21.5 ± 3.7 0.00130.4 ± 4.7 0.008  11 ± 1.0 0.0004  2.5 ± 0.33 <0.0001 TNFSF9 50.8 ± 6.10.0046 20.7 ± 3.1 0.0054  48 ± 2.1 0.99 38.2 ± 6.9  0.334  32 ± 1.120.086 IL-1B  171 ± 9.0 0.0089  187 ± 7.4 0.988 104 ± 21 0.382  88 ± 9.00.204 51.6 ± 5.2  0.033 CXCL1 24.5 ± 0.5 <0.0001 28.2 ± 1.9 0.577 19.8 ±1.8 0.36 12.8 ± 2.5  0.005 5.2 ± 1.0 <0.0001 TNFSF15  9.6 ± 1.1 0.0012 10 ± 1.1 0.998  7.6 ± 0.9 0.544 5.4 ± 0.8 0.053 2.9 ± 0.6 0.003 CCL5 7.6 ± 0.9 0.0045  3.6 ± 0.8 0.26   3 ± 0.5 0.018 2.7 ± 0.2 0.008 2.6 ±1.2 0.006 CCL4 188 ± 12 0.9519 188 ± 16 ns 174 ± 41 ns 191 ± 57  ns 217± 51  ns CCL20  82.5 ± 27.8 0.1189  106 ± 15.1 ns  63 ± 3.1 ns 63.4 ±1.0  ns 42.7 ± 12.7 ns CXCL2  122 ± 11.0 0.9887  125 ± 4.6 ns  128 ±20.0 ns  132 ± 22.9 ns 130 ± 6.4  ns TNF  115 ± 13/1 0.6112 66.4 ± 9.6ns   87 ± 12.4 ns 95.9 ± 21.2 ns  80 ± 14.5 ns BMP6  8.1 ± 1.8 0.1195 4.1 ± 1.1 ns  8.9 ± 1.7 ns 7.8 ± 1.1 ns 3.9 ± 0.5 ns ¹All values arefold change mRNA levels vs. unstimulated PMA-differentiated THP1 cells²Cells were preincubated with 0.4% DMSO or inhibitors for 1 h, thenstimulated with 100 ng/ml LPS for 2 h. ³P-values from one-way ANOVA.⁴P-values from Tukey Honestly Significant Difference post hoc test.

Example 5: Comparing Effects of SB203580 and UM101 on TNFα-Induced GeneExpression in HMVECLs

The effects of UM101 and SB203580 on TNFα-induced gene expression inHMVECLs using RNASeq were compared. HMVECLs were pretreated for 1 h with10 μM SB203580 or 100 μM UM101, and then stimulated with 10 ng/ml TNFαfor 3 h. A UM101 concentration 10-fold higher than its biologicallyeffective dose in HMVECL barrier assays was used, to ensure identifyingany partial overlap with SB203580. The TNFα concentration and durationof stimulation used were based on previously published studies andconfirmed by preliminary qRT-PCR analysis of IL-8 and IL-1ß mRNAexpression (FIG. 4 ). After filtering the RNASeq results for geneshaving ≥10 reads in at least one sample per experiment, 511 genes thatwere upregulated and 520 downregulated by ≥2-fold by TNFα treatment werefound (Table 5).

TABLE 5 RNASeq results filtered for genes with at least 10 reads in onesample per set Log (base 2) fold-change UM101 vs. SB203580 vs. Genecontrol control bin Gene Symbol Gene Name ENSG00000006468 1.146976381.42107626 1 ETV1 ets variant 1 [Source: HGNC Symbol; Acc: HGNC: 3490]ENSG00000128917 1.25167179 1.33201352 1 DLL4 delta-like 4 (Drosophila)[Source: HGNC Symbol; Acc: HGNC: 2910] ENSG00000196872 1.103664091.62514778 1 KIAA1211L KIAA1211-like [Source: HGNC Symbol; Acc: HGNC:33454] ENSG00000108984 1.34524718 1.27752638 1 MAP2K6 mitogen-activatedprotein kinase kinase 6 [Source: HGNC Symbol; Acc: HGNC: 6846]ENSG00000229953 1.63323083 1.70115175 1 RP11-284F21.7 ENSG000002556901.81451563 1.5932836 1 TRIL TLR4 interactor with leucine-rich repeats[Source: HGNC Symbol; Acc: HGNC: 22200] ENSG00000095739 1.113216581.12281058 1 BAMBI BMP and activin membrane- bound inhibitor [Source:HGNC Symbol; Acc: HGNC: 30251] ENSG00000137872 2.48510888 1.90449301 1SEMA6D sema domain, transmembrane domain (TM), and cytoplasmic domain,(semaphorin) 6D [Source: HGNC Symbol; Acc: HGNC: 16770] ENSG000001841851.18112212 1.55055314 1 KCNJ12 potassium channel, inwardly rectifyingsubfamily J, member 12 [Source: HGNC Symbol; Acc: HGNC: 6258]ENSG00000125848 1.25784422 1.46432561 1 FLRT3 fibronectin leucine richtransmembrane protein 3 [Source: HGNC Symbol; Acc: HGNC: 3762]ENSG00000196664 −1.5535936 1.21794414 2 TLR7 toll-like receptor 7[Source: HGNC Symbol; Acc: HGNC: 15631] ENSG00000119714 −1.30406161.22615088 2 GPR68 G protein-coupled receptor 68 [Source: HGNC Symbol;Acc: HGNC: 4519] ENSG00000165379 −1.4439353 2.06145291 2 LRFN5 leucinerich repeat and fibronectin type III domain containing 5 [Source: HGNCSymbol; Acc: HGNC: 20360] ENSG00000135378 −1.1984882 −1.78258053 3 PRRG4proline rich Gla (G- carboxyglutamic acid) 4 (transmembrane) [Source:HGNC Symbol; Acc: HGNC: 30799] ENSG00000145777 −1.3366525 −1.65143959 3TSLP thymic stromal lymphopoietin [Source: HGNC Symbol; Acc: HGNC:30743] ENSG00000102970 −2.7303098 −1.83414345 3 CCL17 chemokine (C-Cmotif) ligand 17 [Source: HGNC Symbol; Acc: HGNC: 10615] ENSG00000259717−1.4952254 −1.6963101 3 LINC00677 long intergenic non-protein coding RNA677 [Source: HGNC Symbol; Acc: HGNC: 20121] ENSG00000205436 −1.160471−1.47916318 3 EXOC3L4 exocyst complex component 3-like 4 [Source: HGNCSymbol; Acc: HGNC: 20120] ENSG00000100985 −1.0911796 −1.15709135 3 MMP9matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa typeIV collagenase) [Source: HGNC Symbol; Acc: HGNC: 7176] ENSG00000131203−3.5679874 −3.51063293 3 IDO1 indoleamine 2,3-dioxygenase 1 [Source:HGNC Symbol; Acc: HGNC: 6059] ENSG00000276408 −3.0213604 −2.36477309 3RP11-490B18.5 ENSG00000169245 −4.3698367 −3.10091556 3 CXCL10 chemokine(C-X-C motif) ligand 10 [Source: HGNC Symbol; Acc: HGNC: 10637]ENSG00000091972 −1.5381554 −1.72928565 3 CD200 CD200 molecule [Source:HGNC Symbol; Acc: HGNC: 7203] ENSG00000110446 −1.7310589 −1.00338842 3SLC15A3 solute carrier family 15 (oligopeptide transporter), member 3[Source: HGNC Symbol; Acc: HGNC: 18068] ENSG00000111424 −1.1973169−1.16718631 3 VDR vitamin D (1,25- dihydroxyvitamin D3) receptor[Source: HGNC Symbol; Acc: HGNC: 12679] ENSG00000125538 −1.1725305−1.40158693 3 IL1B interleukin 1, beta [Source: HGNC Symbol; Acc: HGNC:5992] ENSG00000279805 −1.4538855 −1.28507313 3 CTA-212A2.1ENSG00000202533 −2.8157077 −1.96733528 3 Y_RNA Y RNA [Source: RFAM; Acc:RF00019] ENSG00000181656 −2.1505992 −1.39708375 3 GPR88 Gprotein-coupled receptor 88 [Source: HGNC Symbol; Acc: HGNC: 4539]ENSG00000116031 −3.3824373 −1.54775729 3 CD207 CD207 molecule, langerin[Source: HGNC Symbol; Acc: HGNC: 17935] ENSG00000159450 −1.5476653−1.50495809 3 TCHH trichohyalin [Source: HGNC Symbol; Acc: HGNC: 11791]ENSG00000103044 −1.1243396 −1.43377734 3 HAS3 hyaluronan synthase 3[Source: HGNC Symbol; Acc: HGNC: 4820] ENSG00000225492 −1.7557061−1.3632872 3 GBP1P1 guanylate binding protein 1, interferon-induciblepseudogene 1 [Source: HGNC Symbol; Acc: HGNC: 39561] ENSG00000145113−1.0573157 −2.85949188 3 MUC4 mucin 4, cell surface associated [Source:HGNC Symbol; Acc: HGNC: 7514] ENSG00000164181 −1.3810632 −1.34093337 3ELOVL7 ELOVL fatty acid elongase 7 [Source: HGNC Symbol; Acc: HGNC:26292] ENSG00000169248 −4.1363549 −1.37790594 3 CXCL11 chemokine (C-X-Cmotif) ligand 11 [Source: HGNC Symbol; Acc: HGNC: 10638] ENSG00000162654−2.8359479 −1.25928308 3 GBP4 guanylate binding protein 4 [Source: HGNCSymbol; Acc: HGNC: 20480] ENSG00000144837 −1.5006334 −1.27452433 3 PLA1Aphospholipase A1 member A [Source: HGNC Symbol; Acc: HGNC: 17661]ENSG00000222365 −1.7462552 −2.69898993 3 SNORD12B small nucleolar RNA,C/D box 12B [Source: HGNC Symbol; Acc: HGNC: 33573] ENSG00000237988−3.7376706 −1.08751395 3 OR2I1P olfactory receptor, family 2, subfamilyI, member 1 pseudogene [Source: HGNC Symbol; Acc: HGNC: 8258]ENSG00000163735 −1.4683077 −1.01742785 3 CXCL5 chemokine (C-X-C motif)ligand 5 [Source: HGNC Symbol; Acc: HGNC: 10642] ENSG000002771051.8720451 −1.66179692 4 FP236383.10 ENSG00000259498 1.51730088 0 5RP11-244F12.3 ENSG00000079841 1.1627696 0 5 RIMS1 regulating synapticmembrane exocytosis 1 [Source: HGNC Symbol; Acc: HGNC: 17282]ENSG00000104081 1.87839947 0 5 BMF Bcl2 modifying factor [Source: HGNCSymbol; Acc: HGNC: 24132] ENSG00000128011 1.21234677 0 5 LRFN1 leucinerich repeat and fibronectin type III domain containing 1 [Source: HGNCSymbol; Acc: HGNC: 29290] ENSG00000102760 2.39873086 0 5 RGCC regulatorof cell cycle [Source: HGNC Symbol; Acc: HGNC: 20369] ENSG000002729182.02570082 0 5 CTB-152G17.6 ENSG00000158715 1.09721113 0 5 SLC45A3solute carrier family 45, member 3 [Source: HGNC Symbol; Acc: HGNC:8642] ENSG00000169247 1.22087519 0 5 SH3TC2 SH3 domain andtetratricopeptide repeats 2 [Source: HGNC Symbol; Acc: HGNC: 29427]ENSG00000163235 1.00798575 0 5 TGFA transforming growth factor, alpha[Source: HGNC Symbol; Acc: HGNC: 11765] ENSG00000138311 1.68539518 0 5ZNF365 zinc finger protein 365 [Source: HGNC Symbol; Acc: HGNC: 18194]ENSG00000263426 1.90751532 0 5 RN7SL471P RNA, 7SL, cytoplasmic 471,pseudogene [Source: HGNC Symbol; Acc: HGNC: 46487] ENSG000002038833.1401464 0 5 SOX18 SRY (sex determining region Y)-box 18 [Source: HGNCSymbol; Acc: HGNC: 11194] ENSG00000152213 2.11880858 0 5 ARL11ADP-ribosylation factor-like 11 [Source: HGNC Symbol; Acc: HGNC: 24046]ENSG00000115641 1.20543525 0 5 FHL2 four and a half LIM domains 2[Source: HGNC Symbol; Acc: HGNC: 3703] ENSG00000163884 1.31377126 0 5KLF15 Kruppel-like factor 15 [Source: HGNC Symbol; Acc: HGNC: 14536]ENSG00000171223 1.07792014 0 5 JUNB jun B proto-oncogene [Source: HGNCSymbol; Acc: HGNC: 6205] ENSG00000137875 1.24981 0 5 BCL2L10 BCL2-like10 (apoptosis facilitator) [Source: HGNC Symbol; Acc: HGNC: 993]ENSG00000119630 1.02473901 0 5 PGF placental growth factor [Source: HGNCSymbol; Acc: HGNC: 8893] ENSG00000157404 1.98981566 0 5 KIT v-kitHardy-Zuckerman 4 feline sarcoma viral oncogene homolog [Source: HGNCSymbol; Acc: HGNC: 6342] ENSG00000004799 2.74875752 0 5 PDK4 pyruvatedehydrogenase kinase, isozyme 4 [Source: HGNC Symbol; Acc: HGNC: 8812]ENSG00000104903 1.17436711 0 5 LYL1 lymphoblastic leukemia associatedhematopoiesis regulator 1 [Source: HGNC Symbol; Acc: HGNC: 6734]ENSG00000164683 2.56462289 0 5 HEY1 hes-related family bHLHtranscription factor with YRPW motif 1 [Source: HGNC Symbol; Acc: HGNC:4880] ENSG00000229436 2.97587733 0 5 AC073850.6 ENSG000000745901.86606392 0 5 NUAK1 NUAK family, SNF1-like kinase, 1 [Source: HGNCSymbol; Acc: HGNC: 14311] ENSG00000163121 1.17777496 0 5 NEURL3neuralized E3 ubiquitin protein ligase 3 [Source: HGNC Symbol; Acc:HGNC: 25162] ENSG00000171435 2.0275154 0 5 KSR2 kinase suppressor of ras2 [Source: HGNC Symbol; Acc: HGNC: 18610] ENSG00000225213 2.78814593 0 5RP11- 197M22.2 ENSG00000175556 2.1400816 0 5 LONRF3 LON peptidaseN-terminal domain and ring finger 3 [Source: HGNC Symbol; Acc: HGNC:21152] ENSG00000172031 1.46275504 0 5 EPHX4 epoxide hydrolase 4 [Source:HGNC Symbol; Acc: HGNC: 23758] ENSG00000164284 1.60799521 0 5 GRPEL2GrpE-like 2, mitochondrial (E. coli) [Source: HGNC Symbol; Acc: HGNC:21060] ENSG00000198774 1.92290695 0 5 RASSF9 Ras association(RalGDS/AF-6) domain family (N-terminal) member 9 [Source: HGNC Symbol;Acc: HGNC: 15739] ENSG00000109452 1.25820227 0 5 INPP4B inositolpolyphosphate-4- phosphatase, type II, 105 kDa [Source: HGNC Symbol;Acc: HGNC: 6075] ENSG00000071282 1.2458539 0 5 LMCD1 LIM andcysteine-rich domains 1 [Source: HGNC Symbol; Acc: HGNC: 6633]ENSG00000163545 1.15652631 0 5 NUAK2 NUAK family, SNF1-like kinase, 2[Source: HGNC Symbol; Acc: HGNC: 29558] ENSG00000125968 2.63377664 0 5ID1 inhibitor of DNA binding 1, dominant negative helix- loop-helixprotein [Source: HGNC Symbol; Acc: HGNC: 5360] ENSG000000992601.50498454 0 5 PALMD palmdelphin [Source: HGNC Symbol; Acc: HGNC: 15846]ENSG00000176641 1.2877484 0 5 RNF152 ring finger protein 152 [Source:HGNC Symbol; Acc: HGNC: 26811] ENSG00000139874 1.37617951 0 5 SSTR1somatostatin receptor 1 [Source: HGNC Symbol; Acc: HGNC: 11330]ENSG00000137834 2.51438228 0 5 SMAD6 SMAD family member 6 [Source: HGNCSymbol; Acc: HGNC: 6772] ENSG00000259721 1.0709029 0 5 RP11-758N13.1ENSG00000181800 2.42803687 0 5 CELF2-AS1 CELF2 antisense RNA 1 [Source:HGNC Symbol; Acc: HGNC: 23515] ENSG00000184523 2.14599043 0 5 PTGER4P2prostaglandin E receptor 4 (subtype EP4) pseudogene 2 [Source: HGNCSymbol; Acc: HGNC: 9598] ENSG00000101187 1.12788457 0 5 SLCO4A1 solutecarrier organic anion transporter family, member 4A1 [Source: HGNCSymbol; Acc: HGNC: 10953] ENSG00000237512 1.72226772 0 5 UNC5B-AS1 UNC5Bantisense RNA 1 [Source: HGNC Symbol; Acc: HGNC: 45096] ENSG000001564631.43711148 0 5 SH3RF2 SH3 domain containing ring finger 2 [Source: HGNCSymbol; Acc: HGNC: 26299] ENSG00000137672 1.41739616 0 5 TRPC6 transientreceptor potential cation channel, subfamily C, member 6 [Source: HGNCSymbol; Acc: HGNC: 12338] ENSG00000138135 2.4356406 0 5 CH25Hcholesterol 25-hydroxylase [Source: HGNC Symbol; Acc: HGNC: 1907]ENSG00000183691 1.18113108 0 5 NOG noggin [Source: HGNC Symbol; Acc:HGNC: 7866] ENSG00000139174 2.23155754 0 5 PRICKLE1 prickle homolog 1(Drosophila) [Source: HGNC Symbol; Acc: HGNC: 17019] ENSG000001883051.46592995 0 5 C19orf35 chromosome 19 open reading frame 35 [Source:HGNC Symbol; Acc: HGNC: 24793] ENSG00000082497 3.01359039 0 5 SERTAD4SERTA domain containing 4 [Source: HGNC Symbol; Acc: HGNC: 25236]ENSG00000134215 1.69084 0 5 VAV3 vav 3 guanine nucleotide exchangefactor [Source: HGNC Symbol; Acc: HGNC: 12659] ENSG000002429021.87508823 0 5 RP11-309L24.2 ENSG00000027075 1.03323842 0 5 PRKCHprotein kinase C, eta [Source: HGNC Symbol; Acc: HGNC: 9403]ENSG00000203280 1.22563855 0 5 CTA-221G9.12 ENSG00000006459 1.00029861 05 KDM7A lysine (K)-specific demethylase 7A [Source: HGNC Symbol; Acc:HGNC: 22224] ENSG00000171408 3.15161753 0 5 PDE7B phosphodiesterase 7B[Source: HGNC Symbol; Acc: HGNC: 8792] ENSG00000162981 1.53241836 0 5FAM84A family with sequence similarity 84, member A [Source: HGNCSymbol; Acc: HGNC: 20743] ENSG00000118946 1.93243268 0 5 PCDH17protocadherin 17 [Source: HGNC Symbol; Acc: HGNC: 14267] ENSG000001463761.27126839 0 5 ARHGAP18 Rho GTPase activating protein 18 [Source: HGNCSymbol; Acc: HGNC: 21035] ENSG00000204086 2.05144911 0 5 RPA4replication protein A4, 30 kDa [Source: HGNC Symbol; Acc: HGNC: 30305]ENSG00000221887 1.05893107 0 5 HMSD histocompatibility (minor) serpindomain containing [Source: HGNC Symbol; Acc: HGNC: 23037]ENSG00000196196 1.26354255 0 5 HRCT1 histidine rich carboxyl terminus 1[Source: HGNC Symbol; Acc: HGNC: 33872] ENSG00000172548 2.84971177 0 5NIPAL4 NIPA-like domain containing 4 [Source: HGNC Symbol; Acc: HGNC:28018] ENSG00000156804 2.04515428 0 5 FBXO32 F-box protein 32 [Source:HGNC Symbol; Acc: HGNC: 16731] ENSG00000203684 1.5984677 0 5 IBA57-AS1IBA57 antisense RNA 1 (head to head) [Source: HGNC Symbol; Acc: HGNC:32062] ENSG00000205502 1.63750711 0 5 C2CD4B C2 calcium-dependent domaincontaining 4B [Source: HGNC Symbol; Acc: HGNC: 33628] ENSG000001637341.26141193 0 5 CXCL3 chemokine (C-X-C motif) ligand 3 [Source: HGNCSymbol; Acc: HGNC: 4604] ENSG00000181444 1.48713949 0 5 ZNF467 zincfinger protein 467 [Source: HGNC Symbol; Acc: HGNC: 23154]ENSG00000275342 1.45192287 0 5 SGK223 Tyrosine-protein kinase SgK223[Source: UniProtKB/Swiss- Prot; Acc: Q86YV5] ENSG00000214944 1.431167650 5 ARHGEF28 Rho guanine nucleotide exchange factor (GEF) 28 [Source:HGNC Symbol; Acc: HGNC: 30322] ENSG00000198795 1.37664308 0 5 ZNF521zinc linger protein 521 [Source: HGNC Symbol; Acc: HGNC: 24605]ENSG00000108932 1.95156031 0 5 SLC16A6 solute carrier family 16, member6 [Source: HGNC Symbol; Acc: HGNC: 10927] ENSG00000145990 1.18764084 0 5GFOD1 glucose-fructose oxidoreductase domain containing 1 [Source: HGNCSymbol; Acc: HGNC: 21096] ENSG00000179546 1.7287987 0 5 HTR1D5-hydroxytryptamine (serotonin) receptor 1D, G protein-coupled [Source:HGNC Symbol; Acc: HGNC: 5289] ENSG00000186472 1.73331066 0 5 PCLOpiccolo presynaptic cytomatrix protein [Source: HGNC Symbol; Acc: HGNC:13406] ENSG00000138678 1.55650245 0 5 AGPAT9 1-acylglycerol−3 -phosphateO-acyltransferase 9 [Source: HGNC Symbol; Acc: HGNC: 28157]ENSG00000225814 1.57236046 0 5 GRPEL2P2 GrpE-like 2, mitochondrial (E.coli) pseudogene 2 [Source: HGNC Symbol; Acc: HGNC: 41970]ENSG00000172572 1.01708765 0 5 PDE3A phosphodiesterase 3A,cGMP-inhibited [Source: HGNC Symbol; Acc: HGNC: 8778] ENSG000001072821.0986938 0 5 APBA1 amyloid beta (A4) precursor protein-binding, familyA, member 1 [Source: HGNC Symbol; Acc: HGNC: 578] ENSG000001718771.0590391 0 5 FRMD5 FERM domain containing 5 [Source: HGNC Symbol; Acc:HGNC: 28214] ENSG00000151623 1.83557493 0 5 NR3C2 nuclear receptorsubfamily 3, group C, member 2 [Source: HGNC Symbol; Acc: HGNC: 7979]ENSG00000189184 1.39874706 0 5 PCDH18 protocadherin 18 [Source: HGNCSymbol; Acc: HGNC: 14268] ENSG00000187479 1.56424224 0 5 C11orf96chromosome 11 open reading frame 96 [Source: HGNC Symbol; Acc: HGNC:38675] ENSG00000178726 1.31386114 0 5 THBD thrombomodulin [Source: HGNCSymbol; Acc: HGNC: 11784] ENSG00000137193 2.05607477 0 5 PIM1 Pim-1proto-oncogene, serine/threonine kinase [Source: HGNC Symbol; Acc: HGNC:8986] ENSG00000154734 1.08386589 0 5 ADAMTS1 ADAM metallopeptidase withthrombospondin type 1 motif, 1 [Source: HGNC Symbol; Acc: HGNC: 217]ENSG00000143772 1.05692317 0 5 ITPKB inositol-trisphosphate 3- kinase B[Source: HGNC Symbol; Acc: HGNC: 6179] ENSG00000140022 1.42852098 0 5STON2 stonin 2 [Source: HGNC Symbol; Acc: HGNC: 30652] ENSG000001817221.75771309 0 5 ZBTB20 zinc finger and BTB domain containing 20 [Source:HGNC Symbol; Acc: HGNC: 13503] ENSG00000184058 2.32518737 0 5 TBX1 T-box1 [Source: HGNC Symbol; Acc: HGNC: 11592] ENSG00000043591 1.38727807 0 5ADRB1 adrenoceptor beta 1 [Source: HGNC Symbol; Acc: HGNC: 285]ENSG00000126550 2.94676056 0 5 HTN1 histatin 1 [Source: HGNC Symbol;Acc: HGNC: 5283] ENSG00000143867 1.2361215 0 5 OSR1 odd-skipped relatedtransciption factor 1 [Source: HGNC Symbol; Acc: HGNC: 8111]ENSG00000116833 1.34604824 0 5 NR5A2 nuclear receptor subfamily 5, groupA, member 2 [Source: HGNC Symbol; Acc: HGNC: 7984] ENSG000001662922.00173044 0 5 TMEM100 transmembrane protein 100 [Source: HGNC Symbol;Acc: HGNC: 25607] ENSG00000188487 1.04413181 0 5 INSC inscuteablehomolog (Drosophila) [Source: HGNC Symbol; Acc: HGNC: 33116]ENSG00000176697 1.76616859 0 5 BDNF brain-derived neurotrophic factor[Source: HGNC Symbol; Acc: HGNC: 1033] ENSG00000079102 1.5770273 0 5RUNX1T1 runt-related transcription factor 1; translocated to, 1 (cyclinD-related) [Source: HGNC Symbol; Acc: HGNC: 1535] ENSG000001625991.02633907 0 5 NFIA nuclear factor I/A [Source: HGNC Symbol; Acc: HGNC:7784] ENSG00000188763 1.52543754 0 5 FZD9 frizzled class receptor 9[Source: HGNC Symbol; Acc: HGNC: 4047] ENSG00000154639 1.36734426 0 5CXADR coxsackie virus and adenovirus receptor [Source: HGNC Symbol; Acc:HGNC: 2559] ENSG00000227946 1.24976159 0 5 AC007383.3 ENSG000001433411.16130281 0 5 HMCN1 hemicentin 1 [Source: HGNC Symbol; Acc: HGNC:19194] ENSG00000237892 1.07996952 0 5 KLF7-IT1 KLF7 intronic transcript1 (non-protein coding) [Source: HGNC Symbol; Acc: HGNC: 41355]ENSG00000103522 1.30364536 0 5 IL21R interleukin 21 receptor [Source:HGNC Symbol; Acc: HGNC: 6006] ENSG00000162630 1.457488 0 5 B3GALT2UDP-Gal: betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 2[Source: HGNC Symbol; Acc: HGNC: 917] ENSG00000106069 1.15244395 0 5CHN2 chimerin 2 [Source: HGNC Symbol; Acc: HGNC: 1944] ENSG000001690471.03338349 0 5 IRS1 insulin receptor substrate 1 [Source: HGNC Symbol;Acc: HGNC: 6125] ENSG00000226476 1.25685284 0 5 RP11-776H12.1ENSG00000181016 1.30372056 0 5 LSMEM1 leucine-rich single-pass membraneprotein 1 [Source: HGNC Symbol; Acc: HGNC: 22036] ENSG000001219663.89708969 0 5 CXCR4 chemokine (C-X-C motif) receptor 4 [Source: HGNCSymbol; Acc: HGNC: 2561] ENSG00000189143 1.4778446 0 5 CLDN4 claudin 4[Source: HGNC Symbol; Acc: HGNC: 2046] ENSG00000257642 2.54048396 0 5RP11-474B16.1 ENSG00000250271 2.3827306 0 5 RP11-64D22.5 ENSG000001884831.76997593 0 5 IER5L immediate early response 5- like [Source: HGNCSymbol; Acc: HGNC: 23679] ENSG00000183775 1.04020782 0 5 KCTD16potassium channel tetramerization domain containing 16 [Source: HGNCSymbol; Acc: HGNC: 29244] ENSG00000107984 1.55122779 0 5 DKK1 dickkopfWNT signaling pathway inhibitor 1 [Source: HGNC Symbol; Acc: HGNC: 2891]ENSG00000174514 0 1.18746783 6 MFSD4 major facilitator superfamilydomain containing 4 [Source: HGNC Symbol; Acc: HGNC: 25433]ENSG00000270379 0 1.09045988 6 HEATR9 HEAT repeat containing 9 [Source:HGNC Symbol; Acc: HGNC: 26548] ENSG00000240859 0 1.36188025 6AC093627.10 ENSG00000236671 0 1.65509644 6 PRKG1-AS1 PRKG1 antisense RNA1 [Source: HGNC Symbol; Acc: HGNC: 45029] ENSG00000261707 0 1.15736629 6RP11- 264M12.2 ENSG00000273669 0 3.69876021 6 RP11- 405M12.4ENSG00000231345 0 1.35868618 6 BEND3P1 BEN domain containing 3pseudogene 1 [Source: HGNC Symbol; Acc: HGNC: 45014] ENSG00000134253 01.10224143 6 TRIM45 tripartite motif containing 45 [Source: HGNC Symbol;Acc: HGNC: 19018] ENSG00000138336 0 1.76426632 6 TET1 tet methylcytosinedioxygenase 1 [Source: HGNC Symbol; Acc: HGNC: 29484] ENSG00000120162 01.23378866 6 MOB3B MOB kinase activator 3B [Source: HGNC Symbol; Acc:HGNC: 23825] ENSG00000171860 0 1.01814888 6 C3AR1 complement component3a receptor 1 [Source: HGNC Symbol; Acc: HGNC: 1319] ENSG00000167676 01.03160807 6 PLIN4 perilipin 4 [Source: HGNC Symbol; Acc: HGNC: 29393]ENSG00000237234 0 1.66084244 6 RP1-142L7.5 ENSG00000164124 0 1.053810286 TMEM144 transmembrane protein 144 [Source: HGNC Symbol; Acc: HGNC:25633] ENSG00000118513 0 1.12982301 6 MYB v-myb avian myeloblastosisviral oncogene homolog [Source: HGNC Symbol; Acc: HGNC: 7545]ENSG00000091137 0 1.16699806 6 SLC26A4 solute carrier family 26 (anionexchanger), member 4 [Source: HGNC Symbol; Acc: HGNC: 8818]ENSG00000198483 0 1.43532228 6 ANKRD35 ankyrin repeat domain 35 [Source:HGNC Symbol; Acc: HGNC: 26323] ENSG00000237886 0 1.57281997 6 LINC01573long intergenic non-protein coding RNA 1573 [Source: HGNC Symbol; Acc:HGNC: 51192] ENSG00000174004 0 2.27602232 6 NRROS negative regulator ofreactive oxygen species [Source: HGNC Symbol; Acc: HGNC: 24613]ENSG00000185634 0 1.66433678 6 SHC4 SHC (Src homology 2 domaincontaining) family, member 4 [Source: HGNC Symbol; Acc: HGNC: 16743]ENSG00000259886 0 1.17666464 6 ENSG00000145358 0 1.32550219 6 DDIT4LDNA-damage-inducible transcript 4-like [Source: HGNC Symbol; Acc: HGNC:30555] ENSG00000269896 0 1.28838255 6 RP4-740C4.5 ENSG00000135828 01.0837094 6 RNASEL ribonuclease L (2′,5′- oligoisoadenylate synthetase-dependent) [Source: HGNC Symbol; Acc: HGNC: 10050] ENSG00000259162 01.52667158 6 RP11-203M5.6 ENSG00000279109 −3.3403535 0 7 AC008641.1Uncharacterized protein {ECO: 0000313|Ensembl: EN SP00000485568}[Source: UniProtKB/TrEMBL; Acc: A0A096LPF4] ENSG00000152778 −1.0972226 07 IFIT5 interferon-induced protein with tetratricopeptide repeats 5[Source: HGNC Symbol; Acc: HGNC: 13328] ENSG00000128284 −1.3050716 0 7APOL3 apolipoprotein L, 3 [Source: HGNC Symbol; Acc: HGNC: 14868]ENSG00000213886 −4.0396114 0 7 UBD ubiquitin D [Source: HGNC Symbol;Acc: HGNC: 18795] ENSG00000164116 −1.5223055 0 7 GUCY1A3 guanylatecyclase 1, soluble, alpha 3 [Source: HGNC Symbol; Acc: HGNC: 4685]ENSG00000137462 −1.0020489 0 7 TLR2 toll-like receptor 2 [Source: HGNCSymbol; Acc: HGNC: 11848] ENSG00000049249 −2.1008906 0 7 TNFRSF9 tumornecrosis factor receptor superfamily, member 9 [Source: HGNC Symbol;Acc: HGNC: 11924] ENSG00000169181 −2.095598 0 7 GSG1L GSG1-like [Source:HGNC Symbol; Acc: HGNC: 28283] ENSG00000162888 −1.7912286 0 7 C1orf147chromosome 1 open reading frame 147 [Source: HGNC Symbol; Acc: HGNC:32061] ENSG00000107201 −1.2413536 0 7 DDX58 DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 58 [Source: HGNC Symbol; Acc: HGNC: 19102] ENSG00000179826−2.5133806 0 7 MRGPRX3 MAS-related GPR, member X3 [Source: HGNC Symbol;Acc: HGNC: 17980] ENSG00000132109 −1.2867007 0 7 TRIM21 tripartite motifcontaining 21 [Source: HGNC Symbol; Acc: HGNC: 11312] ENSG00000215007−1.0078729 0 7 DNAJA1P3 DnaJ (Hsp40) homolog, subfamily A, member 1pseudogene 3 [Source: HGNC Symbol; Acc: HGNC: 39339] ENSG00000204682−1.1628447 0 7 CASC10 cancer susceptibility candidate 10 [Source: HGNCSymbol; Acc: HGNC: 31448] ENSG00000108688 −1.8703428 0 7 CCL7 chemokine(C-C motif) ligand 7 [Source: HGNC Symbol; Acc: HGNC: 10634]ENSG00000112096 −1.0730214 0 7 SOD2 superoxide dismutase 2,mitochondrial [Source: HGNC Symbol; Acc: HGNC: 11180] ENSG00000010379−2.697306 0 7 SLC6A13 solute carrier family 6 (neurotransmittertransporter), member 13 [Source: HGNC Symbol; Acc: HGNC: 11046]ENSG00000169403 −1.579388 0 7 PTAFR platelet-activating factor receptor[Source: HGNC Symbol; Acc: HGNC: 9582] ENSG00000115604 −3.2590591 0 7IL18R1 interleukin 18 receptor 1 [Source: HGNC Symbol; Acc: HGNC: 5988]ENSG00000133401 −1.024655 0 7 PDZD2 PDZ domain containing 2 [Source:HGNC Symbol; Acc: HGNC: 18486] ENSG00000095587 −2.2510173 0 7 TLL2tolloid-like 2 [Source: HGNC Symbol; Acc: HGNC: 11844] ENSG00000134256−1.3577477 0 7 CD101 CD101 molecule [Source: HGNC Symbol; Acc: HGNC:5949] ENSG00000272463 −1.1413394 0 7 RP11-532F6.3 ENSG00000102794−1.5942464 0 7 IRG1 immunoresponsive 1 homolog (mouse) [Source: HGNCSymbol; Acc: HGNC: 33904] ENSG00000223799 −1.6341444 0 7 IL10RB-AS1IL10RB antisense RNA 1 (head to head) [Source: HGNC Symbol; Acc: HGNC:44303] ENSG00000019582 −1.1055825 0 7 CD74 CD74 molecule, majorhistocompatibility complex, class II invariant chain [Source: HGNCSymbol; Acc: HGNC: 1697] ENSG00000121577 −1.2357302 0 7 POPDC2 popeyedomain containing 2 [Source: HGNC Symbol; Acc: HGNC: 17648]ENSG00000215268 −1.7373145 0 7 LA16c-60G3.8 ENSG00000119121 −1.4785767 07 TRPM6 transient receptor potential cation channel, subfamily M, member6 [Source: HGNC Symbol; Acc: HGNC: 17995] ENSG00000108576 −1.2299055 0 7SLC6A4 solute carrier family 6 (neurotransmitter transporter), member 4[Source: HGNC Symbol; Acc: HGNC: 11050] ENSG00000274818 −1.8854991 0 7RP1-292L20.3 ENSG00000198133 −1.9389698 0 7 TMEM229B transmembraneprotein 229B [Source: HGNC Symbol; Acc: HGNC: 20130] ENSG00000130477−1.2541053 0 7 UNC13A unc-13 homolog A (C. elegans) [Source: HGNCSymbol; Acc: HGNC: 23150] ENSG00000266094 −1.0579637 0 7 RASSF5 Rasassociation (RalGDS/AF-6) domain family member 5 [Source: HGNC Symbol;Acc: HGNC: 17609] ENSG00000137571 −1.0904993 0 7 SLCO5A1 solute carrierorganic anion transporter family, member 5A1 [Source: HGNC Symbol; Acc:HGNC: 19046] ENSG00000272512 −1.5915514 0 7 RP11-54O7.17 ENSG00000124391−1.7278326 0 7 IL17C interleukin 17C [Source: HGNC Symbol; Acc: HGNC:5983] ENSG00000136052 −1.3272223 0 7 SLC41A2 solute carrier family 41(magnesium transporter), member 2 [Source: HGNC Symbol; Acc: HGNC:31045] ENSG00000185245 −1.9464332 0 7 GP1BA glycoprotein Ib (platelet),alpha polypeptide [Source: HGNC Symbol; Acc: HGNC: 4439] ENSG00000203685−1.8061183 0 7 C1orf95 chromosome 1 open reading frame 95 [Source: HGNCSymbol; Acc: HGNC: 30491] ENSG00000149654 −1.331613 0 7 CDH22 cadherin22, type 2 [Source: HGNC Symbol; Acc: HGNC: 13251] ENSG00000230943−1.574129 0 7 RP11-367G18.1 ENSG00000215277 −3.2333936 0 7 RNF212B ringfinger protein 212B [Source: HGNC Symbol; Acc: HGNC: 20438]ENSG00000112139 −1.2861961 0 7 MDGA1 MAM domain containingglycosylphosphatidylinositol anchor 1 [Source: HGNC Symbol; Acc: HGNC:19267] ENSG00000143494 −1.5135205 0 7 VASH2 vasohibin 2 [Source: HGNCSymbol; Acc: HGNC: 25723] ENSG00000151883 −1.1760751 0 7 PARP8 poly(ADP-ribose) polymerase family, member 8 [Source: HGNC Symbol; Acc:HGNC: 26124] ENSG00000136514 −1.9750242 0 7 RTP4 receptor (chemosensory)transporter protein 4 [Source: HGNC Symbol; Acc: HGNC: 23992]ENSG00000106258 −1.012592 0 7 CYP3A5 cytochrome P450, family 3,subfamily A, polypeptide 5 [Source: HGNC Symbol; Acc: HGNC: 2638]ENSG00000243649 −2.5551714 0 7 CFB complement factor B [Source: HGNCSymbol; Acc: HGNC: 1037] ENSG00000164342 −1.0290951 0 7 TLR3 toll-likereceptor 3 [Source: HGNC Symbol; Acc: HGNC: 11849] ENSG00000115956−2.3479537 0 7 PLEK pleckstrin [Source: HGNC Symbol; Acc: HGNC: 9070]ENSG00000144476 −1.7184658 0 7 ACKR3 atypical chemokine receptor 3[Source: HGNC Symbol; Acc: HGNC: 23692] ENSG00000157601 −1.4173764 0 7MX1 MX dynamin-like GTPase 1 [Source: HGNC Symbol; Acc: HGNC: 7532]ENSG00000177409 −1.1465499 0 7 SAMD9L sterile alpha motif domaincontaining 9-like [Source: HGNC Symbol; Acc: HGNC: 1349] ENSG00000119917−1.8565474 0 7 IFIT3 interferon-induced protein with tetratricopeptiderepeats 3 [Source: HGNC Symbol; Acc: HGNC: 5411] ENSG00000271503−1.7683442 0 7 CCL5 chemokine (C-C motif) ligand 5 [Source: HGNC Symbol;Acc: HGNC: 10632] ENSG00000117226 −1.1447048 0 7 GBP3 guanylate bindingprotein 3 [Source: HGNC Symbol; Acc: HGNC: 4184] ENSG00000163840−1.3311379 0 7 DTX3L deltex 3 like, E3 ubiquitin ligase [Source: HGNCSymbol; Acc: HGNC: 30323] ENSG00000010030 −1.2207673 0 7 ETV7 etsvariant 7 [Source: HGNC Symbol; Acc: HGNC: 18160] ENSG00000261884−1.3310986 0 7 CTC-479C5.12 Uncharacterized protein {ECO:0000313|Ensembl: ENSP00000463376} [Source: UniProtKB/TrEMBL; Acc:J3QL48] ENSG00000152229 −1.0179291 0 7 PSTPIP2 proline-serine-threoninephosphatase interacting protein 2 [Source: HGNC Symbol; Acc: HGNC: 9581]ENSG00000100678 −2.0514071 0 7 SLC8A3 solute carrier family 8(sodium/calcium exchanger), member 3 [Source: HGNC Symbol; Acc: HGNC:11070] ENSG00000225194 −2.4201688 0 7 LINC00092 long intergenicnon-protein coding RNA 92 [Source: HGNC Symbol; Acc: HGNC: 31408]ENSG00000140968 −1.1898419 0 7 IRF8 interferon regulatory factor 8[Source: HGNC Symbol; Acc: HGNC: 5358] ENSG00000006210 −1.2182721 0 7CX3CL1 chemokine (C-X3-C motif) ligand 1 [Source: HGNC Symbol; Acc:HGNC: 10647] ENSG00000221963 −1.1392138 0 7 APOL6 apolipoprotein L, 6[Source: HGNC Symbol; Acc: HGNC: 14870] ENSG00000130589 −1.0673283 0 7HELZ2 helicase with zinc finger 2, transcriptional coactivator [Source:HGNC Symbol; Acc: HGNC: 30021] ENSG00000239713 −1.6622438 0 7 APOBEC3Gapolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G[Source: HGNC Symbol; Acc: HGNC: 17357] ENSG00000151023 −1.0139189 0 7ENKUR enkurin, TRPC channel interacting protein [Source: HGNC Symbol;Acc: HGNC: 28388] ENSG00000187123 −1.3193979 0 7 LYPD6 LY6/PLAUR domaincontaining 6 [Source: HGNC Symbol; Acc: HGNC: 28751] ENSG00000253831−3.0507137 0 7 ETV3L ets variant 3-like [Source: HGNC Symbol; Acc: HGNC:33834] ENSG00000246130 −3.0351799 0 7 RP11-875O11.2 ENSG00000128335−1.8122079 0 7 APOL2 apolipoprotein L, 2 [Source: HGNC Symbol; Acc:HGNC: 619] ENSG00000108702 −4.9466198 0 7 CCL1 chemokine (C-C motif)ligand 1 [Source: HGNC Symbol; Acc: HGNC: 10609] ENSG00000105963−1.2049889 0 7 ADAP1 ArfGAP with dual PH domains 1 [Source: HGNC Symbol;Acc: HGNC: 16486] ENSG00000170075 −1.4491156 0 7 GPR37L1 Gprotein-coupled receptor 37 like 1 [Source: HGNC Symbol; Acc: HGNC:14923] ENSG00000267607 −1.2021234 0 7 CTD-2369P2.8 ENSG00000142961−1.1266143 0 7 MOB3C MOB kinase activator 3C [Source: HGNC Symbol; Acc:HGNC: 29800] ENSG00000159200 −1.1629133 0 7 RCAN1 regulator ofcalcineurin 1 [Source: HGNC Symbol; Acc: HGNC: 3040] ENSG00000185291−1.5031919 0 7 IL3RA interleukin 3 receptor, alpha (low affinity)[Source: HGNC Symbol; Acc: HGNC: 6012] ENSG00000135917 −1.3131434 0 7SLC19A3 solute carrier family 19 (thiamine transporter), member 3[Source: HGNC Symbol; Acc: HGNC: 16266] ENSG00000179817 −1.7239398 0 7MRGPRX4 MAS-related GPR, member X4 [Source: HGNC Symbol; Acc: HGNC:17617] ENSG00000173918 −1.1809051 0 7 C1QTNF1 C1q and tumor necrosisfactor related protein 1 [Source: HGNC Symbol; Acc: HGNC: 14324]ENSG00000198879 −1.3578392 0 7 SFMBT2 Scm-like with four mbt domains 2[Source: HGNC Symbol; Acc: HGNC: 20256] ENSG00000272078 −1.1495108 0 7RP4-734G22.3 ENSG00000269794 −1.7476941 0 7 AC010642.2 ENSG00000115919−1.2162129 0 7 KYNU kynureninase [Source: HGNC Symbol; Acc: HGNC: 6469]ENSG00000255521 −1.8970116 0 7 RP4-607I7.1 ENSG00000173193 −1.3401257 07 PARP14 poly (ADP-ribose) polymerase family, member 14 [Source: HGNCSymbol; Acc: HGNC: 29232] ENSG00000183644 −1.6099016 0 7 C11orf88chromosome 11 open reading frame 88 [Source: HGNC Symbol; Acc: HGNC:25061] ENSG00000253522 −1.2658724 0 7 CTC-231O11.1 ENSG00000236453−1.8969845 0 7 AC003092.1 ENSG00000131979 −1.5762138 0 7 GCH1 GTPcyclohydrolase 1 [Source: HGNC Symbol; Acc: HGNC: 4193] ENSG00000069493−2.1742062 0 7 CLEC2D C-type lectin domain family 2, member D [Source:HGNC Symbol; Acc: HGNC: 14351] ENSG00000069696 −1.1323561 0 7 DRD4dopamine receptor D4 [Source: HGNC Symbol; Acc: HGNC: 3025]ENSG00000175356 −1.3984803 0 7 SCUBE2 signal peptide, CUB domain,EGF-like 2 [Source: HGNC Symbol; Acc: HGNC: 30425] ENSG00000128165−1.1779326 0 7 ADM2 adrenomedullin 2 [Source: HGNC Symbol; Acc: HGNC:28898] ENSG00000166856 −1.1992337 0 7 GPR182 G protein-coupled receptor182 [Source: HGNC Symbol; Acc: HGNC: 13708] ENSG00000199161 −1.616849 07 MIR126 microRNA 126 [Source: HGNC Symbol; Acc: HGNC: 31508]ENSG00000050730 −1.9873165 0 7 TNIP3 TNFAIP3 interacting protein 3[Source: HGNC Symbol; Acc: HGNC: 19315] ENSG00000255750 −1.8909724 0 7RP11-283G6.5 ENSG00000184530 −2.3505853 0 7 C6orf58 chromosome 6 openreading frame 58 [Source: HGNC Symbol; Acc: HGNC: 20960] ENSG00000104883−1.3860147 0 7 PEX11G peroxisomal biogenesis factor 11 gamma [Source:HGNC Symbol; Acc: HGNC: 20208] ENSG00000129521 −2.7016498 0 7 EGLN3egl-9 family hypoxia- inducible factor 3 [Source: HGNC Symbol; Acc:HGNC: 14661] ENSG00000204482 −1.2475769 0 7 LST1 leukocyte specifictranscript 1 [Source: HGNC Symbol; Acc: HGNC: 14189] ENSG00000115267−1.3445539 0 7 IFIH1 interferon induced with helicase C domain 1[Source: HGNC Symbol; Acc: HGNC: 18873] ENSG00000162692 −2.1801821 0 7VCAM1 vascular cell adhesion molecule 1 [Source: HGNC Symbol; Acc: HGNC:12663] ENSG00000261618 −1.2864344 0 7 RP11-79H23.3 ENSG00000101276−1.1705916 0 7 SLC52A3 solute carrier family 52 (riboflavintransporter), member 3 [Source: HGNC Symbol; Acc: HGNC: 16187]ENSG00000064309 −1.4651234 0 7 CDON cell adhesion associated, oncogeneregulated [Source: HGNC Symbol; Acc: HGNC: 17104] ENSG00000167371−1.4282411 0 7 PRRT2 proline-rich transmembrane protein 2 [Source: HGNCSymbol; Acc: HGNC: 30500] ENSG00000101017 −1.5583663 0 7 CD40 CD40molecule, TNF receptor superfamily member 5 [Source: HGNC Symbol; Acc:HGNC: 11919] ENSG00000164400 0 −1.63408281 8 CSF2 colony stimulatingfactor 2 (granulocyte-macrophage) [Source: HGNC Symbol; Acc: HGNC: 2434]ENSG00000172602 0 −1.15027449 8 RND1 Rho family GTPase 1 [Source: HGNCSymbol; Acc: HGNC: 18314] ENSG00000174502 0 −2.09273487 8 SLC26A9 solutecarrier family 26 (anion exchanger), member 9 [Source: HGNC Symbol; Acc:HGNC: 14469] ENSG00000234290 0 −1.00067832 8 AC116366.6 ENSG000001709610 −2.64619793 8 HAS2 hyaluronan synthase 2 [Source: HGNC Symbol; Acc:HGNC: 4819] ENSG00000110848 0 −1.46706866 8 CD69 CD69 molecule [Source:HGNC Symbol; Acc: HGNC: 1694] ENSG00000164512 0 −1.20441285 8 ANKRD55ankyrin repeat domain 55 [Source: HGNC Symbol; Acc: HGNC: 25681]ENSG00000167034 0 −1.10541745 8 NKX3-1 NK3 homeobox 1 [Source: HGNCSymbol; Acc: HGNC: 7838] ENSG00000105246 0 −1.39394774 8 EBI3Epstein-Barr virus induced 3 [Source: HGNC Symbol; Acc: HGNC: 3129]ENSG00000145506 0 −1.41815829 8 NKD2 naked cuticle homolog 2(Drosophila) [Source: HGNC Symbol; Acc: HGNC: 17046] ENSG00000127533 0−2.64266235 8 F2RL3 coagulation factor II (thrombin) receptor-like 3[Source: HGNC Symbol; Acc: HGNC: 3540] ENSG00000115008 0 −2.15272028 8IL1A interleukin 1, alpha [Source: HGNC Symbol; Acc: HGNC: 5991]ENSG00000073282 0 −1.31215479 8 TP63 tumor protein p63 [Source: HGNCSymbol; Acc: HGNC: 15979] ENSG00000113196 0 −1.61798433 8 HAND1 heartand neural crest derivatives expressed 1 [Source: HGNC Symbol; Acc:HGNC: 4807] ENSG00000096996 0 −1.40936482 8 IL12RB1 interleukin 12receptor, beta 1 [Source: HGNC Symbol; Acc: HGNC: 5971] ENSG000002755820 −1.05575947 8 RP4-681N20.5 ENSG00000244476 0 −1.23492596 8 ERVFRD-1endogenous retrovirus group FRD, member 1 [Source: HGNC Symbol; Acc:HGNC: 33823] ENSG00000165685 0 −1.10987961 8 TMEM52B transmembraneprotein 52B [Source: HGNC Symbol; Acc: HGNC: 26438] ENSG00000172331 0−1.20450079 8 BPGM 2,3-bisphosphoglycerate mutase [Source: HGNC Symbol;Acc: HGNC: 1093] ENSG00000198846 0 −1.42254609 8 TOX thymocyteselection- associated high mobility group box [Source: GNC Symbol; Acc:HGNC: 18988] ENSG00000258521 0 −1.0229865 8 RP11-638I2.9 ENSG000002791330 −1.46999903 8 RP11-342K2.1 ENSG00000121905 0 −2.21994573 8 HPCAhippocalcin [Source: HGNC Symbol; Acc: HGNC: 5144] ENSG00000232810 0−1.4782116 8 TNF tumor necrosis factor [Source: HGNC Symbol; Acc: HGNC:11892] ENSG00000178882 0 −1.71201963 8 FAM101A family with sequencesimilarity 101, member A [Source: HGNC Symbol; Acc: HGNC: 27051]ENSG00000173391 0 −1.01327133 8 OLR1 oxidized low density lipoprotein(lectin-like) receptor 1 [Source: HGNC Symbol; Acc: HGNC: 8133]ENSG00000257671 0 −1.03664909 8 RP3-416H24.1 ENSG00000269826 0−1.64046441 8 RP11-158I3.3 ENSG00000176907 0 −1.2182476 8 C8orf4chromosome 8 open reading frame 4 [Source: HGNC Symbol; Acc: HGNC: 1357]ENSG00000165478 0 −1.21164831 8 HEPACAM hepatic and glial cell adhesionmolecule [Source: HGNC Symbol; Acc: HGNC: 26361] ENSG00000175746 0−1.57742953 8 C15orf54 chromosome 15 open reading frame 54 [Source: HGNCSymbol; Acc: HGNC: 33797] ENSG00000187848 0 −2.24446361 8 P2RX2purinergic receptor P2X, ligand gated ion channel, 2 [Source: HGNCSymbol; Acc: HGNC: 15459] List of genes modified by SB203580 or UM101(see also FIG. 5); Bin number refers to gene expression responsepattern: 1 = increased expression with both inhibitors 5 = increasedwith UM101, unchanged with SB203580 2 = decreased with UM101, increasedwith SB203580 6 = unchanged with UM101, increased with SB203580 3 =decreased expression with both inhibitors 7 = decreased with UM101,unchanged with SB203580 4 = increased with UM101, decreased withSB203580 8 = unchanged with UM101, decreased with SB203580

SB203580 inhibited expression of 61 TNFα-induced genes, 28 of which werealso inhibited by UM101 (Table 6, Table 5, FIG. 5 ). SB203580 increasedexpression of 38 genes, 10 of which were also increased by UM101. Of the28 genes inhibited by both SB203580 and UM101, 22 coded for knownproteins, including IL-1ß, CCL17, MMNP9, IDO1, CXCL5, 10 and 11,hyaluronan synthase-3, MUC4, and PLA2 (Table 6). Of the 33 genesinhibited by SB203580 but not UM101, 24 coded for known proteins,including GM-CSF, IL-1α, TNFα, IL-12 receptor-β1, and hyaluronansynthase-2 (Table 6).

TABLE 6 Effect of SB203580 and UM101 in HMVECLs on TNFα-induced genes¹Gene LOG fold-change LOG fold-change Symbol Gene name SB203580 vs. DMSOUM101 vs. DMSO Genes inhibited by both SB203580 and UM101 PRRG4 prolinerich Gla 4 −1.782580534 −1.198488233 TSLP thymic stromal lymphopoietin−1.651439594 −1.336652511 CCL17 chemokine (C-C motif) ligand 17−1.834143455 −2.730309773 EXOC3L4 exocyst complex component 3-like 4−1.479163179 −1.160471021 MMP9 matrix metallopeptidase 9 −1.157091348−1.091179627 IDO1 indoleamine 2,3-dioxygenase 1 −3.510632932−3.567987354 CXCL10 chemokine (C-X-C motif) ligand 10 −3.100915562−4.369836708 CD200 CD200 −1.729285649 −1.538155406 SLC15A3 solutecarrier family 15, member 3 −1.00338842 −1.73105887 VDR Vitamin Dreceptor −1.16718631 −1.19731694 IL1B Interleukin-1β −1.401586926−1.172530543 GPR88 G protein-coupled receptor 88 −1.397083754−2.150599176 CD207 CD207 (langerin) −1.547757288 −3.382437255 TCHHtrichohyalin −1.504958085 −1.547665316 HAS3 hyaluronan synthase 3−1.43377734 −1.124339564 GBP1P1 guanylate binding protein 1 −1.363287203−1.755706078 MUC4 Mucin-4 −2.859491876 −1.057315692 ELOVL7 ELOVL fattyacid elongase 7 −1.340933369 −1.381063226 CXCL11 chemokine (C-X-C motif)ligand 11 −1.377905942 −4.136354868 GBP4 guanylate binding protein 4−1.259283076 −2.835947907 PLA1A phospholipase A1 member A −1.27452433−1.500633356 CXCL5 chemokine (C-X-C motif) ligand 5 −1.017427849−1.468307731 Genes inhibited by SB203580 but not UM101 CSF2 GM-CSF−1.634082807 ns² RND1 Rho family GTPase 1 −1.15027449 ns SLC26A9 solutecarrier family 26, member 9 −2.092734866 ns HAS2 hyaluronan synthase 2−2.646197932 ns CD69 CD69 −1.467068659 ns ANKRD55 ankyrin repeat domain55 −1.204412851 ns NKX3-1 NK3 homeobox 1 −1.105417452 ns EBI3Epstein-Barr virus induced 3 −1.393947741 ns NKD2 naked cuticle homolog2 −1.418158287 ns F2RL3 coagulation factor II receptor-like 3−2.642662346 ns ILIA Interleukin-1alpha −2.152720278 ns TP63 Tumorprotein 63 −1.312154792 ns HAND1 heart and neural crest derivatives−1.617984328 ns expressed 1 IL12RB1 interleukin 12 receptor, beta 1−1.409364824 ns ERVFRD-1 endogenous retrovirus group FRD, −1.234925956ns member 1 TMEM52B transmembrane protein 52B −1.109879612 ns BPGM2,3-bisphosphoglycerate mutase −1.204500786 ns TOX thymocyteselection-associated high −1.422546092 ns mobility group box HPCAhippocalcin −2.219945733 ns TNF Tumor necrosis factor-alpha −1.478211598ns FAM101A family with sequence similarity 101, −1.712019627 ns member AOLR1 oxidized low density lipoprotein −1.013271327 ns receptor 1 HEPACAMhepatic and glial cell adhesion molecule −1.211648309 ns P2RX2purinergic receptor P2X −2.244463614 ns ¹HMVECLs were preincubated witheither 0.4% DMSO, 10 μM SB20350, or 100 μM UM101 for 1 h, thenstimulated with 10 ng/ml TNFα for 4 h and RNASeq performed. ²notsignificant

The differentially expressed genes were further analyzed usingPathwayNet and Ingenuity™ tools to identify the transcription factorsand biological pathways regulated by the two inhibitors. PathwayNetanalysis suggested that UM101 inhibits some of the SB203580-inhibitedtranscription factors (Stat-1, c-Fos, c-Jun, NFκB, p53, PPARγ, and Sp1),but not others (ATF1, ATF2, Elk1, c/EBPβ, USF1, SMAD3, FOXO1, and CREBvia MSK1/2). Ingenuity™ analysis suggested that both SB203580 and UM101inhibit the Dendritic Cell Maturation, Triggering Receptor Expressed onMyeloid cells-1 (TREM1), High Mobility Group Box 1 (HMGB1), and NFκBpathways and both increase Liver X-Receptor/Retinoid X-Receptor(LXR/RXR) activation, while only SB203580 inhibits IL-6, Acute Phase,and Cholecystokinin/Gastrin-mediated pathways (FIG. 3A). UM101 at 100 μMreduced expression of 115 genes and increased expression of 119 genesthat were not modified by SB203580 (Table 5), which Ingenuity™ pathwayanalysis suggested reduced Toll-like receptor and Wnt/β-cateninsignaling and increased Nitric Oxide in Cardiovascular Disease pathways(FIG. 3B).

Example 6: Comparing Effects of SB203580 and UM101 on p38 MAPK SubstratePhosphorylation Profile

To assess whether UM101 selectively inhibits phosphorylation consistentwith its target, HeLa cells were pretreated for 30 min with 10 μMSB203580, 50 μM UM101, or 0.1% DMSO vehicle control, then with the p38activator, anisomycin (25 μg/ml) and phosphorylated MK2, and Stat-1 wereanalyzed by immunoblotting (FIG. 3C). Anisomycin-stimulatedphosphorylation of MK2 and Stat-1 were reduced by both 10 μM SB203580and 50 μM UM1010, but more so with SB203580.

Example 7: Analyzing Specific Binding of UM101 to p38α

DSF was used to analyze concentration-specific binding of UM101 to p38αand p38ß. While SB203580 stabilized both p38α and p38β, UM101 onlystabilized p38α (FIG. 3D). To confirm that UM101 bound the CADD-targetedpocket, DSF was used to compare UM101- and SB203580-binding to wild-typep388a and a p388a mutant with four of the ten target pocket amino acids(R49K/HL107-8TF/K165R) substituted (FIG. 3E). The mutant exhibitedSB203580-binding that was identical to wild-type p38α, but noUM101-binding.

Selective binding of UM101 to the CADD-targeted pocket in p38α wasconfirmed using Saturation Transfer Difference (STD)-NMR. A 1D spectrumof UM101 in the presence of p38α is shown in FIG. 3F and the STDspectrum of the same sample is shown in FIG. 3G. The peaks in the 1Dspectrum are labeled according to tentative peak assignments of UM101 inaqueous form, based on assignments of UM101 in 2 mM d6-DMSO, which wereobtained from the use of 1D proton and C13 and 2D-HMBC experiments. Theshifts of the peaks in the STD spectrum correspond well to those of the1D spectrum, thus indicating that protons in both aromatic rings ofUM101 interact with p38α. In contrast, while the 1D spectra for UM101with p38β and mutated p38α were similar to that of UM101/p38α (FIG. 3Hand FIG. 3I), the interaction of UM101 with p38β and mutated p38α ismuch weaker, as indicated by the barely discernible peaks of thearomatic protons in the STD spectrum of UM101 with p38β (FIG. 3I) andmutated p38α (FIG. 3K).

Example 8: Synthetic Methods for Preparing Exemplary Compounds of theInvention

General Methods for Chemistry: All air or moisture sensitive reactionswere performed under positive pressure of nitrogen with oven-driedglassware. Chemical reagents and anhydrous solvents are obtained fromcommercial sources and used as-is.

The p38α MAPK inhibitors of the invention can be prepared by methodsgenerally known in the art. For example, compound UM101 can be preparedas depicted in Scheme 1. UM101 can be prepared in two steps from threecommercially available fragments (Scheme 1), which facilitates itsoptimization. Acylation of 4-aminobenzaldehyde with 4-chlorobenzoylchloride in the presence of diisopropylamine (DIPEA) generates anintermediate aldehyde. Subsequent reductive amination of the aldehydewith thiomorpholine 1,1-dioxide and Na-triacetoxyborohydride(NaBH(OAc)₃) affords UM101.

A focused structure-activity relationship (SAR) of UM101 and additionallead compounds is conducted to determine its pharmacophore and theinformation used to achieve its optimization, for example by feedingthis information back into the CADD model to improve its predictabilitythereby facilitating subsequent design cycles. A summary of the proposedmodifications to UM101 is shown in Scheme 2, which is driven by theSILCS molecular modeling, and addresses improvements in binding affinityand specificity, and enhancements in physicochemical properties.Importantly, the STD-NMR analysis of UM101 confirmed that both itsaromatic rings interact with the protein, hence modification of theserings will impact binding affinities. First, since thepositively-charged (under physiological conditions) piperidine-typenitrogen of UM101 is predicted to interact with negatively-chargedresidues such as D112 and D168, will be retained in some embodiments.According to the FragMaps, aliphatic (e.g., cyclohexyl) or aromatic(e.g. furan) substituents off the central phenyl ring should enhancebinding to the protein via interactions with V30, V38, A51, 184, L108,and L167. In addition, the presence of hydrogen bond acceptor mapsoverlapping with the aliphatic maps suggest that combinedaliphatic/hydrogen bond acceptor groups, such as OEt, are incorporatedortho to the aniline nitrogen in some embodiments. Hydrogen bond donor(e.g. NH₂, OH) and/or acceptor groups (e.g. OMe, isoxazole) areincorporated into the ortho and meta positions of the peripheralchlorophenyl ring in some embodiments, which may be replaced with achloropyridyl ring in other embodiments. These changes further increasecompound solubility. Although the chlorine of the chlorophenyl ringinteracts modestly with the protein as judged by SILCS GFE analysis insome embodiments, this site is also varied with alternative hydrophobicand more polar groups in other embodiments. The trans-amide bond ismodified to a rigid E-alkene in some embodiments, and a more flexiblesulfonamide in other embodiments. The sulfone S02 group contributes −0.5kcal/mol to binding based on SILCS GFE analysis, indicating that thisregion of the molecule can be exploited to optimize the molecule'sphysicochemical properties without compromising binding affinity in someembodiments. For example, the S02 group is replaced with a polar oxygenatom in some embodiments, and also an NMe group in other embodiments.

Example 9: Novel Non-ATP/Catalytic Site p38 Mitogen Activated ProteinKinase Inhibitors

Computer-aided drug design (CADD) was used to identify novel smallmolecule compounds that target select substrate-binding sites andinhibit p38 mitogen activated protein (MAP) kinase activity thatcontributes to acute lung injury. These compounds are unique in thatthey are NON-ATP/catalytic site kinase inhibitors and provide analternative to the current p38 MAP kinase inhibitors that target ATPbinding or catalytic sites and block all enzyme activity. Compound UM101targets the p38α MAP kinase isoform and is the major p38 MAP kinaseisoform associated with chronic inflammation and tissue damageassociated with acute lung injury. Current p38 MAP kinase inhibitorstarget the ATP binding or catalytic site, which blocks all enzymeactivity, and lack isoform selectivity. In contrast, the compoundsdisclosed herein target sites that are unique to protein substrateinteractions and block specific p38α MAP kinase functions.

Novel UM101 analogues were synthesized (Schemes 3-5) and tested forinteractions with p38α MAP kinase and biological activity.

Two lines of evidence suggest that the lead compounds can improve lungbarrier function and prevent lung injury. First, the compounds can blockthrombin-induced cell permeability in human vascular endothelial cells(FIG. 8 ). Second, mice challenged with LPS to induce lung inflammationhad reduced protein leak into the bronchoaveolar lavage fluid in thepresence of the parent compound UM101 and analogues SF-6-222 andSF-7-009 (FIG. 9 ), or analogues SF-6-222, SF-7-009, and SF-7-044 (FIG.10 ). As shown in FIG. 9 , UM101 and analogues inhibit acute lung injuryin a mouse model. Mice were pretreated i.p. with 1 mg of test compoundsand then treated with 100 mg LPS i.t. at 39° C. to induce inflammation.Lung injury was assessed by measuring total protein in the lung lavagefluid. As shown in FIG. 10 , UM101 and analogues inhibit acute lunginjury in a mouse model. All mice received 50 μg LPS i.t. and were kept24 h at 39° C.; all treatments 1 mg in 0.5 ml DMSO given 6 h post-LPS;mean±SM, n=5, * p=0.03 vs DMSO #1; † p=0.16 vs DMSO #2. Lung injury wasassessed by measuring total protein in the lung lavage fluid.

The useful application of this invention is to provide new therapies foracute lung injury and other diseases caused by inflammation andendothelial permeability. In addition, the invention is aimed atovercoming issues with toxicity and poor efficacy with the current p38MAP kinase inhibitors used in the clinic to treat inflammation.

A number of patent and non-patent publications are cited herein in orderto describe the state of the art to which this invention pertains. Theentire disclosure of each of these publications is incorporated byreference herein.

While certain embodiments of the present invention have been describedand/or exemplified above, various other embodiments will be apparent tothose skilled in the art from the foregoing disclosure. The presentinvention is, therefore, not limited to the particular embodimentsdescribed and/or exemplified, but is capable of considerable variationand modification without departure from the scope and spirit of theappended claims.

Moreover, as used herein, the term “about” means that amounts, sizes,formulations, parameters, shapes and other quantities andcharacteristics are not and need not be exact, but may be approximateand/or larger or smaller, as desired, reflecting tolerances, conversionfactors, rounding off, measurement error and the like, and other factorsknown to those of skill in the art. In general, an amount, size,formulation, parameter, shape or other quantity or characteristic is“about” or “approximate” whether or not expressly stated to be such.

Furthermore, the transitional terms “comprising”, “consistingessentially of” and “consisting of”, when used in the appended claims,in original and amended form, define the claim scope with respect towhat unrecited additional claim elements or steps, if any, are excludedfrom the scope of the claim(s). The term “comprising” is intended to beinclusive or open-ended and does not exclude any additional, unrecitedelement, method, step or material. The term “consisting of” excludes anyelement, step or material other than those specified in the claim and,in the latter instance, impurities ordinary associated with thespecified material(s). The term “consisting essentially of” limits thescope of a claim to the specified elements, steps or material(s) andthose that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. All compounds, compositions,formulations, and methods described herein that embody the presentinvention can, in alternate embodiments, be more specifically defined byany of the transitional terms “comprising,” “consisting essentially of,”and “consisting of.”

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1. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein, L¹ is selectedfrom —CH₂—, —C(CH₃)₂—, and —C(CH₂CH₂)—; L² is selected from —NH—SO₂—,—NHCH₂—, —CH₂NH—, —NHCO—, —CONH, and —SO₂NH—; and each of R^(1a) andR^(2a) is independently selected from hydrogen, C₁₋₁₀ alkyl, and C₁₋₁₀alkyl substituted by one or more of substituents which are independentlyhydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro,trimethylsilanyl, —OR^(a), —SR^(a), —S(O)_(t)R^(a) (wherein t is 1 or2), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, —N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (wherein t is 1 or 2), —S(O)_(t)R^(a) (wherein tis 1 or 2), —S(O)_(t)OR^(a) (wherein t is 1 or 2), —S(O)_(t)N(R^(a))₂(wherein t is 1 or 2), or —PO(OR^(a))₂ wherein each R^(a) isindependently hydrogen and C₁₋₃ alkyl.
 2. The compound of claim 1,wherein L¹ is —CH₂—.
 3. The compound of claim 1, wherein L² is —NH—SO₂—.4. The compound of claim 1, wherein L² is —NHCH₂—.
 5. The compound ofclaim 1, wherein L² is —CH₂NH—.
 6. The compound of claim 1, wherein L²is —NHCO—.
 7. The compound of claim 1, wherein L² is —CONH—.
 8. Thecompound of claim 1, wherein L² is —SO₂—NH—.
 9. The compound of claim 1,wherein each of R^(1a) and R^(2a) is independently selected fromhydrogen and C₁₋₃ alkyl.
 10. The compound of claim 1, wherein each ofR^(1a) and R^(2a) is independently C₁₋₃ alkyl.
 11. A pharmaceuticalcomposition comprising the compound of claim 1 or a pharmaceuticallyacceptable salt thereof.
 12. The pharmaceutical composition of claim 11,wherein the pharmaceutical composition is an oral pharmaceuticalcomposition.
 13. The pharmaceutical composition of claim 11, wherein thepharmaceutical composition is an oral dosage form.
 14. A method oftreating an inflammatory disease in a patient comprising administeringto a patient in need thereof a therapeutically effective amount of thecompound of claim 1 or a pharmaceutically acceptable salt thereof,wherein the inflammatory disease is selected from rheumatoid arthritis,a cardiovascular disease, multiple sclerosis, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), asthma, acuterespiratory distress syndrome (ARDS), and acute lung injury (ALI). 15.The method of claim 14, wherein the disease is a respiratory disease.16. The method of claim 14, wherein administering comprises orallyadministering.
 17. The method of claim 14, wherein administeringcomprises administering an oral dosage form comprising the compound. 18.The method of claim 14, wherein administering comprises administeringfrom 0.1 mg/kg to 200 mg/kg of the compound.